Modular dna-binding domains and methods of use

ABSTRACT

The present invention refers to methods for selectively recognizing a base pair in a DNA sequence by a polypeptide, to modified polypeptides which specifically recognize one or more base pairs in a DNA sequence and, to DNA which is modified so that it can be specifically recognized by a polypeptide and to uses of the polypeptide and DNA in specific DNA targeting as well as to methods of modulating expression of target genes in a cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 13/016,297,filed Jan. 28, 2011 which is a continuation of International ApplicationPCT/IB2010/000154, filed Jan. 12, 2010, which designates the U.S and waspublished by the International Bureau in English on Jul. 15, 2010, andwhich claims the benefit of U.S. Provisional Patent Application No.61/225,043, filed Jul. 12, 2009, European (EP) Patent Applicaiton No.09165328.7, filed Jul. 13, 2009, German (DE) Patent Application No.102009004659.3, filed Jan. 12, 2009; all of which are herebyincorporated herein in their entirety by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention refers to methods for selectively recognizing abase pair in a target DNA sequence by a polypeptide, to modifiedpolypeptides which specifically recognize one or more base pairs in atarget DNA sequence and, to DNA which is modified so that it can bespecifically recognized by a polypeptide and to uses of the polypeptideand DNA in specific DNA targeting as well as to methods of modulatingexpression of target genes in a cell.

BACKGROUND OF THE INVENTION

Phytopathogenic bacteria of the genus Xanthomonas cause severe diseaseson many important crop plants. The bacteria translocate an arsenal ofeffectors including members of the large transcription activator-like(TAL)/AvrBs3-like effector family via the type III secretion system intoplant cells (Kay & Bonas (2009)Curr. Opin. Microbiol. 12:37-43, White &Yang (2009) Plant Physiol. doi:10.1104/pp. 1109.139360; Schornack et al.(2006) J. Plant Physiol. 163:256-272). TAL effectors, key virulencefactors of Xanthomonas, contain a central domain of tandem repeats,nuclear localization signals (NLSs), and an activation domain (AD) andact as transcription factors in plant cells (Kay et al. (2007)Science318:648-651; Römer et al. (2007) Science 318:645-648; Gu et al.(2005)Nature 435, 1122-1125; FIG. 1 a). The type member of this effectorfamily, AvrBs3 from Xanthomonas campestris pv. vesicatoria, contains17.5 repeats and induces expression of UPA (upregulated by AvrBs3) genesincluding the Bs3 resistance gene in pepper plants (Kay et al.(2007)Science 318:648-651; Römer et al. (2007)Science 318:645-648;Marois et al. (2002)Mol. Plant-Microbe Interact. 15:637-646). The numberand order of repeats in a TAL effector determine its specific activity(Herbers et al. (1992) Nature 356:172-174). The repeats were shown to beessential for DNA-binding of AvrBs3 and constitute a novel DNA-bindingdomain (Kay et al. (2007)Science 318:648-651). How this domain contactsDNA and what determines specificity has remained enigmatic.

Selective gene expression is mediated via the interaction of proteintranscription factors with specific nucleotide sequences within theregulatory region of the gene. The manner in which DNA-binding proteindomains are able to discriminate between different DNA sequences is animportant question in understanding crucial processes such as thecontrol of gene expression in differentiation and development.

The ability to specifically design and generate DNA-binding domains thatrecognize a desired DNA target is highly desirable in biotechnology.Such ability can be useful for the development of custom transcriptionfactors with the ability to modulate gene expression upon target DNAbinding. Examples include the extensive work done with the design ofcustom zinc finger DNA-binding proteins specific for a desired targetDNA sequence (Choo et al. (1994) Nature 372:645; Pomerantz et al.,(1995) Science 267:93-96; Liu et al., Proc. Natl. Acad. Sci. USA94:5525-5530 (1997); Guan et al. (2002)Proc. Natl. Acad. Sci. USA99:13296-13301; U.S. Pat. No. 7,273,923; U.S. Pat. No. 7,220,719).Furthermore, polypeptides containing designer DNA-binding domains can beutilized to modify the actual target DNA sequence by the inclusion ofDNA modifying domains, such as a nuclease catalytic domain, within thepolypeptide. Examples of such include the DNA binding domain of ameganuclease/homing endonuclease DNA recognition site in combinationwith a non-specific nuclease domain (see US Pat. Appl. 2007/0141038),modified meganuclease DNA recognition site and/or nuclease domains fromthe same or different meganucleases (see U.S. Pat. App. Pub.20090271881), and zinc finger domains in combination with a domain withnuclease activity, typically from a type IIS restriction endonucleasesuch as FokI (Bibikova et al. (2003)Science 300:764; Urnov et al.(2005)Nature 435, 646; Skukla, et al. (2009) Nature 459, 437-441;Townsend et al. (2009)Nature 459:442445; Kim et al. (1996) Proc. Natl.Acad. Sci. USA 93:1156-1160; U.S. Pat. No. 7,163,824). The currentmethods utilized for identifying custom zinc finger DNA-binding domainsemploy combinatorial selection-based methods utilizing large randomizedlibraries (typically >10⁸ in size) to generate multi-finger domains withdesired DNA specificity (Greisman & Pabo (1997) Science 275:657-661;Hurt et al. (2003)Proc Natl Acad Sci USA 100:12271-12276; Isalan et al.(2001)Nat Biotechnol 19:656-660. Such methods are time intensive,technically demanding and potentially quite costly. The identificationof a simple recognition code for the engineering of DNA-bindingpolypeptides would represent a significant advancement over the currentmethods for designing DNA-binding domains that recognize a desirednucleotide target.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method for producing a polypeptide thatselectively recognizes a base pair in a DNA sequence, the methodcomprising synthesizing a polypeptide comprising a repeat domain,wherein the repeat domain comprises at least one repeat unit derivedfrom a transcription activator-like (TAL) effector, wherein the repeatunit comprises a hypervariable region which determines recognition of abase pair in the DNA sequence, wherein the repeat unit is responsiblefor the recognition of one base pair in the DNA sequence. Thesepolypeptides of the invention comprise repeat units of the presentinvention and can be constructed by a modular approach by preassemblingrepeat units in target vectors that can subsequently be assembled into afinal destination vector. The invention provides the polypeptideproduced the this method as well as DNA sequences encoding thepolypeptides and host organisms and cells comprising such DNA sequences.

The present invention provides a method for selectively recognizing abase pair in a target DNA sequence by a polypeptide wherein saidpolypeptide comprises at least a repeat domain comprising repeat unitswherein in said repeat units each comprise a hypervariable region whichdetermines recognition of a base pair in said target DNA sequence.

More specifically, the inventors have determined those amino acids in aDNA-binding polypeptide responsible for selective recognition of basepairs in a target DNA sequence. With elucidation of the recognitioncode, a general principle for recognizing specific base pairs in atarget DNA sequence by selected amino acids in a polypeptide has beendetermined. The inventors have found that distinct types of repeat unitsthat are part of a repeat unit array of varying length have the capacityto recognize one defined/specific base pair. Within each repeat unitforming a repeat domain, a hypervariable region is responsible for thespecific recognition of a base pair in a target DNA sequence.

Thus, the present invention provides not only a method for selectivelyrecognizing a base pair in a target DNA sequence by a polypeptidecomprising at least a repeat domain comprising repeat units but alsomethods wherein target DNA sequences can be generated which areselectively recognized by repeat domains in a polypeptide.

The invention also provides for a method for constructing polypeptidesthat recognize specific DNA sequences. These polypeptides of theinvention comprise repeat units of the present invention and can beconstructed by a modular approach by preassembling repeat units intarget vectors that can subsequently be assembled into a finaldestination vector.

The invention also provides a method for targeted modulation of geneexpression by constructing modular repeat units specific for a targetDNA sequence of interest, modifying a polypeptide by the addition ofsaid repeat units so as to enable said polypeptide to now recognize thetarget DNA, introducing or expressing said modified polypeptide in aprokaryotic or eurkaryotic cell so as to enable said modifiedpolypeptide to recognize the target DNA sequence, and modulation of theexpression of said target gene in said cell as a result of suchrecognition.

The invention also provides a method for directed modification of atarget DNA sequence by the construction of a polypeptide including atleast a repeat domain of the present invention that recognizes saidtarget DNA sequence and that said polypeptide also contains a functionaldomain capable of modifying the target DNA (such as via site specificrecombination, restriction or integration of donor target sequences)thereby enabling targeted DNA modifications in complex genomes.

The invention further provides for the production of modifiedpolypeptides including at least a repeat domain comprising repeat unitswherein a hypervariable region within each of the repeat unitsdetermines selective recognition of a base pair in a target DNAsequence.

In a further embodiment of the invention, DNA is provided which encodesfor a polypeptide containing a repeat domain as described above.

In a still further embodiment of the invention, DNA is provided which ismodified to include one or more base pairs located in a target DNAsequence so that said each of the base pairs can be specificallyrecognized by a polypeptide including a repeat domain havingcorresponding repeat units, each repeat unit comprising a hypervariableregion which determines recognition of the corresponding base pair insaid DNA.

In a still further embodiment of the invention, uses of thosepolypeptides and DNAs are provided. Additionally provided are plants,plant parts, seeds, plant cells and other non-human host cellstransformed with the isolated nucleic acid molecules of the presentinvention and the proteins or polypeptides encoded by the codingsequences of the present invention. Still further, the polypeptides andDNA described herein can be introduced into animal and human cells aswell as cells of other organisms like fungi or plants.

In summary, the invention focuses on a method for selectivelyrecognizing base pairs in a target DNA sequence by a polypeptide whereinsaid polypeptide comprises at least a repeat domain comprising repeatunits wherein each repeat unit contains a hypervariable region whichdetermines recognition of a base pair in said target DNA sequencewherein consecutive repeat units correspond to consecutive base pairs insaid target DNA sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Model for DNA-target specificity of TAL effectors.

(A) TAL effectors contain central tandem repeat units (red), nuclearlocalization signals (NLS) and an activation domain (AD). Amino acidsequence of the first repeat of AvrBs3. Hypervariable amino acids 12 and13 are shaded in gray.(B) Hypervariable amino acids at position 12 and 13 of the 17.5 AvrBs3repeat units are aligned to the UPA-box consensus (21).(C) Repeat units of TAL effectors and predicted target sequences inpromoters of induced genes were aligned manually. Nucleotides in theupper DNA strand that correspond to the hypervariable amino acids ineach repeat were counted based on the following combinations of eighteffectors and experimentally identified target genes: AvrBs3/Bs3, UPA10,UPA12, UPA14, UPA19, UPA20, UPA21, UPA23, UPA25, AvrBs3Δrep16/Bs3-E,AvrBs3Δrep109/Bs3, AvrHah1/Bs3, AvrXa27/Xa27, PthXo1/Xa13,PthXo6/OsTFX1, PthXo7/OsTFIIAγ1 (see FIG. 5). Predominant combinations(n>4) are shaded in gray. An asterisk indicates that amino acid 13 ismissing in this repeat type.(D) DNA target specificity code (R=A/G; N=A/C/G/T) of repeat types basedon the hypervariable amino acids 12 and 13 (experimentally proven inthis study).

FIG. 2. Target DNA sequences of Hax2, Hax3, and Hax4.

(A) Amino acids 12 and 13 of the Hax2, Hax3, and Hax4 repeat units andpredicted target DNA specificities (Hax-box).(B) Hax-boxes were cloned in front of the minimal Bs4 promoter into aGUS reporter vector.(C) Specific inducibility of the Hax-boxes by Hax effectors. GUSreporter constructs were codelivered via A. tumefaciens into N.benthamiana with 35S-driven hax2, hax3, hax4, and empty T-DNA (−),respectively (error bars indicate SD; n=3 samples; 4-MU,4-methyl-umbelliferone). 35S::uidA (+) served as control. Leaf discswere stained with X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide).

FIG. 3. DNA base pair recognition specificities of repeat types.

(A) Hax4- and ArtX-box-derivatives were cloned in front of the minimalBs4 promoter into a GUS reporter vector.(B) Specificity of NG-, HD-, NI-, and NS-repeat units. Hax4-inducibilityof Hax4-box derivatives permutated in repeat type target bases (graybackground).(C) Specificity of NN-repeat units. Artificial effector ArtX1 andpredicted target DNA sequences. ArtX1-inducibility of ArtX1 boxderivatives permutated in NN-repeat target bases (gray background).(D) Artificial effectors ArtX2 and ArtX3 and derived DNA targetsequences.(E) Specific inducibility of ArtX-boxes by artificial effectors.(A)-(E) GUS reporter constructs were co-delivered via A. tumefaciensinto N. benthamiana with 35S-driven hax4, artX1, artX2, or artX3 genes,and empty T-DNA (−), respectively. 35S::uidA (+) served as control. Leafdiscs were stained with X-Gluc. For quantitative data see FIG. 11.

FIG. 4. A minimal number of repeat units is required for transcriptionalactivation.

(A) Artificial ArtHD effectors with different numbers (0.5-15.5) ofHD-repeat units (total 1.5 to 16.5 repeat units).(B) An ArtHD target box consisting of TA and 17 C was cloned in front ofthe minimal Bs4 promoter into a GUS reporter vector.(C) Promoter activation by ArtHD effectors with different number ofrepeat units. 35S-driven effector gene or empty T-DNA (−) werecodelivered via A. tumefaciens with the GUS-reporter construct into N.benthamiana (error bars indicate SD; n=3 samples; 4-MU). 35S::uidA (+)served as control. Leaf discs were stained with X-Gluc.

FIG. 5. Alignment of DNA target sequences in promoters of induced geneswith the hypervariable amino acids 12 and 13 of TAL effector repeatunits.

(A) Repeat units of AvrBs3, AvrBs3Δrep16, AvrBs3Δrep109, and AvrHah1were aligned to the UPA-box in the promoter of the pepper ECW-30R Bs3gene (accession: EU078684). AvrBs3Δrep16 and AvrBs3Δrep109 are deletionderivatives of AvrBs3 in which repeat units 11-14 and repeat units 12-14were deleted, respectively. AvrBs3, AvrBs3Δrep109, and AvrHah1, but notAvrBs3Δrep16 induce the HR in ECW-30R plants.(B) Repeat units of AvrBs3, AvrBs3Δrep16, AvrBs3Δrep109, and AvrHah1were aligned to the non-functional UPA-box in the promoter of the pepperECW Bs3-E gene (accession: EU078683). AvrBs3Δrep16, but not AvrBs3,AvrBs3Δrep109, or AvrHah1 induce the HR in pepper ECW plants.(C) Repeat units of AvrXa27 were aligned to a putative target sequencein the promoter of the rice Xa27 gene. Xa27 (accession: AY986492) isinduced by AvrXa27 in rice cultivar IRBB27 leading to an HR, but notxa27 (accession: AY986491) in rice cultivar IR24.(D) Repeat units of PthXo1 were aligned to a putative target sequence inthe promoter of the rice Xa13/Os8N3 gene. Xa13 (accession: DQ421396) isinduced by PthXo1 in rice cultivar IR24 leading to susceptibility, butnot xal3 (accession: DQ421394) in rice cultivar IRBB13.(E) Repeat units of PthXo6 were aligned to a putative target sequence inthe promoter of the rice OsTFX1 gene (accession: AK108319). OsTFX1 isinduced by PthXo6 in rice cultivar IR24.(F) Repeat units of PthXo7 were aligned to a putative target sequence inthe promoter of the rice OsTFIIAγ1 gene (CB097192). OsTFIIAγ1 is inducedby PthXo7 in rice cultivar IR24.(A)-(F) Numbers above the DNA sequences indicate nucleotide distance tothe first ATG in the coding region. Repeat/base combinations notmatching our predicted target specificity (amino acids 12/13: NI=A;HD=C; NG=T; NS=A/C/G/T; NN=A/G; IG=T) are coloured in red. Repeat unitswith unknown target DNA specificity are coloured in green.

FIG. 6. The DNA region protected by AvrBs3Δrep16 is 4 bp shorter thanwith AvrBs3.

Summary of DNaseI footprint analyses with AvrBs3 and AvrBs3Δrep16 (seeFIGS. 7, 8).

(A) Bs3 (top) and Bs3-E (middle) promoter sequences protected by AvrBs3and AvrBs3Δrep16, respectively. DNaseI footprinting revealed that AvrBs3protected 37 nucleotides of the sense strand and 36 nucleotides of theantisense strand of the Bs3 promoter, and AvrBs3Δrep16 protected 30nucleotides of the sense strand and 32 nucleotides of the antisensestrand of the Bs3-E promoter. The UPA-box and the predictedAvrBs3Δrep16-box are underlined. UPA20-ubm-r16 (lower part) promotersequences protected by AvrBs3 and AvrBs3Δrep16. The UPA20-ubmr16promoter is a UPA20 promoter derivative with a 2 bp substitution (GA toCT, bold italic) that results in recognition by both, AvrBs3 andAvrBs3Δrep16. DNaseI footprinting revealed that 35 nucleotides of thesense strand and 34 nucleotides of the antisense strand are protected byAvrBs3 (UPA-box is underlined), and 31 nucleotides of the sense strandand 32 nucleotides of the antisense strand are protected by AvrBs3Δrep16(AvrBs3Δrep16-box is underlined). DNA regions shaded in green (AvrBs3)or red (AvrBs3Δrep16) refer to the core footprints which were protectedby AvrBs3 and AvrBs3Δrep16, respectively, in every experiment, even withlow protein amounts (equal molarity of DNA and protein dimers). DNAregions shaded in gray refer to nucleotides which were not protected inall of the 4 experiments at all protein concentrations by the givenproteins. Please note that the 5′ ends of the AvrBs3- andAvrBs3Δrep16-protected regions are identical. Dashed vertical linesindicate the differences between the 3′ ends of the AvrBs3- andAvrBs3Δrep16-protected promoter regions which corroborates our modelthat one repeat contacts one base pair in the DNA.(B) Alignment of AvrBs3 and AvrBs3Δrep16 target DNA sequences in theUPA20-ubm-r16 promoter with AvrBs3 and AvrBs3Δrep16 repeat regions(hypervariable amino acids at position 12 and 13). Repeat/basecombinations not matching our predicted target specificity (amino acids12/13: NI=A; HD=C; NG=T; NS=A/C/G/T) are coloured in red.

FIG. 7. Bs3 and Bs3-E promoter sequences protected by AvrBs3 andAvrBs3Δrep16, respectively.

A representative DNaseI footprint experiment is shown. AvrBs3 DNaseIfootprint on the Bs3 promoter sequence (A, upper/sense DNA strand; B,lower/antisense DNA strand). AvrBs3Δrep16 DNaseI footprint on the Bs3-Epromoter sequence (C, upper, sense DNA strand; D, lower antisense DNAstrand).

(A)-(D) (top) Fluorescently labelled PCR product was incubated with a 5×molar excess (calculated for protein dimers) of His6::AvrBs3,His6::AvrBs3Δrep16, and BSA, respectively, treated with DNaseI andanalyzed on a capillary sequencer. The y axis of the electropherogramshows the relative fluorescence intensity corresponding to the5′-6-FAM-labelled sense strand (a, c) or the 5′-HEX-labelled antisensestrand (b, d) of the PCR product on an arbitrary scale. The traces forthe reactions with His6::AvrBs3 (green) or His 6::AvrBs3Δrep16 (red),respectively, and BSA (black, negative control) were superimposed. Areduction of peak height in the presence of AvrBs3 or AvrBs3Δrep16,respectively, in comparison to the negative control corresponds toprotection. The protected region is indicated by green (AvrBs3) or red(AvrBs3Δrep16) vertical lines, (middle) Electropherogram of the DNAsequence. Orange coloured peaks with numbers correspond to the DNAnucleotide size standard. The predicted target boxes of the effectors inthe DNA sequence are underlined. Nucleotides covered are marked by agreen (AvrBs3) or red (AvrBs3Δrep16) box. Numbers below refer tonucleotide positions relative to the transcription start (+1) in thepresence of AvrBs3 (a, b) or AvrBs3Δrep16 (c, d), respectively. (bottom)DNA PCR product used for DNaseI footprinting, amplified from the Bs3 (a,b) or Bs3-E (c, d) promoters, respectively. The protected regions on thesingle DNA strands are indicated by gray boxes. Numbers below refer tonucleotide positions relative to the transcription start (+1) in thepresence of AvrBs3 (a, b) or AvrBs3Δrep16 (c, d), respectively. Theexperiments were repeated three times with similar results.

FIG. 8. UPA20-ubm-r16 promoter sequence protected by AvrBs3 andAvrBs3Δrep16.

A representative DNaseI footprint experiment. AvrBs3 and AvrBs3Δrep16DNaseI footprint on the UPA20-ubm-r16 promoter sequence (A), upper,sense DNA strand; (B) lower, antisense DNA strand). (top Fluorescentlylabelled PCR product was incubated with a 5× molar excess ofHis6::AvrBs3, His6::AvrBs3Δrep16 and BSA (calculated for proteindimers), respectively, treated with DNaseI and analyzed on a capillarysequencer. The y axis of the electropherogram shows the relativefluorescence intensity corresponding to the 5′-6-FAM-labelled sensestrand (a) or the 5′-HEX-labelled antisense strand (b) of the PCRproduct on an arbitrary scale. The traces for the reactions withHis6::AvrBs3 (green), His6::AvrBs3Δrep16 (red) and the negative controlBSA (black) were superimposed. A reduction of peak height in thepresence of AvrBs3 and AvrBs3Δrep16 in comparison to the negativecontrol corresponds to protection. The protected regions are indicatedby green (AvrBs3) and red (AvrBs3Δrep16) vertical lines. (middle)Electropherogram of the DNA sequence. Orange coloured peaks with numberscorrespond to the DNA nucleotide size standard. Nucleotides covered byAvrBs3 are marked by green lines and a green box (with the UPA boxunderlined), nucleotides covered by AvrBs3Δrep16 are marked by red linesand a red box (with the AvrBs3Δrep16-box underlined). The UPA20-ubm-r16mutation (GA to CT) is indicated in italics. (bottom) DNA PCR productused for DNaseI footprinting, amplified from the UPA20-ubm-r16 promoter.The protected regions on the single DNA strands are indicated by grayboxes. Numbers below refer to nucleotide positions relative to thetranscription start (+1) of the UPA20 wildtype promoter in the presenceof AvrBs3. The experiment was repeated three times with similar results.

FIG. 9. GUS reporter constructs.

Target DNA sequences (TAL effector-box) were inserted 5′ of the minimaltomato Bs4 promoter (41) (pBs4; −50 to +25) sequence and transferred byGATEWAY recombination into the A. tumefaciens T-DNA vector pGWB330constructing a fusion to a promoterles uidA (β-glucuronidase, GUS) gene.attB1, attB2; GATEWAY recombination sites.

FIG. 10. Recognition specificity of the putative repeat 0 in Hax3.

(A) Amino acids 12 and 13 of Hax3-repeat units and four possible targetHax3-boxes with permutations in the position corresponding to repeat 0.(B) The target boxes were cloned in front of the minimal tomato Bs4promoter into a GUS reporter vector.(C) GUS activities with 35S-driven hax3 or empty T-DNA (−) codeliveredvia A. tumefaciens with the GUS reporter constructs into N. benthamianaleaf cells (4-MU, 4-methyl-umbelliferone; n=3; error bars indicate SD).For qualitative assays, leaf discs were stained with X-Gluc. Theexperiment was performed twice with similar results.

FIG. 11. DNA base pair recognition specificities of repeat types.

Hax4- and ArtX-box-derivatives were cloned in front of the minimal Bs4promoter into a GUS reporter vector. Quantitative data to FIG. 3.

(A) Specificity of NG-, HD-, NI-, and NS-repeat units. Hax4-inducibilityof Hax4-box derivatives permutated in repeat type target bases.(B) Specificity of NN-repeat units. ArtX1-inducibility of ArtX1-boxderivatives permutated in NN-repeat target bases.(C) Specific inducibility of ArtX-boxes by artificial effectors ArtX1,ArtX2, and ArtX3, respectively.

(A)-(C) GUS reporter constructs were codelivered via A. tumefaciens intoN. benthamiana leaf cells together with 35S-driven hax4, artX1, artX2,artX3 genes (gray bars), and empty T-DNA (a, b, white bars; c, -),respectively (n=3; error bars indicate SD). 35S::uidA (+) served ascontrol. The experiments were performed three times with similarresults.

FIG. 12. Predicted target DNA sequences for AvrXa10.

(A) Amino acids 12 and 13 of the AvrXa10-repeat units and two possibletarget boxes with predicted NN type repeat-specificity A or G.(B) AvrXa10 target boxes were cloned in front of the minimal Bs4promoter into a GUS reporter vector.(C) GUS assay of 35S-driven avrXa10, hax3 (specificity control), orempty T-DNA (−) codelivered via A. tumefaciens with GUS reporterconstructs into N. benthamiana leaf cells. 35S::uidA (+) served asconstitutive control (n=3; error bars indicate SD). For qualitativeassays, leaf discs were stained with X-Gluc. The experiment wasperformed three times with similar results.

FIG. 13. Recognition specificity of the repeat type IG in Hax2.

(A) Amino acids 12 and 13 of Hax2 repeat units and four possible targetHax2-boxes for repeat type IG.(B) The Hax2 target boxes were cloned in front of the minimal Bs4promoter into a GUS reporter vector.(C) GUS assay of 35S promoter-driven hax2 or empty T-DNA (−) codeliveredvia A. tumefaciens with the GUS reporter constructs into N. benthamianaleaf cells. 35S::uidA (+) served as constitutive control (n=3; errorbars indicate SD. For qualitative assays, leaf discs were stained withX-Gluc. The experiment was performed three times with similar results.

FIG. 14. Hax2 induces expression of PAP1 in A. thaliana.

(A) Leaves of A. thaliana were inoculated with A. tumefaciens strainsdelivering T-DNA constructs for 35S-driven expression of hax2, hax3, andhax4, respectively. Expression of hax2, but not of hax3 and hax4 inducedpurple pigmentation suggestive of anthocyanin production. The photographwas taken 7 days post inoculation.(B) Transgenic A. thaliana line carrying hax2 under control of anethanol-inducible promoter. Plants of a segregating T2 population weresprayed with 10% ethanol to induce expression of the transgene. Onlyhax2-transgenic plants accumulated anthocyanin. The photograph was taken6 days post treatment.(C) Semiquantitative RT-PCR of hax2 (29 cycles), PAP1 (32 cycles), andelongation factor Tu (EF-Tu, 32 cycles) with cDNA from hax2-transgenicplants of three independent A. thaliana lines before (−) and 24 h after(+) spraying with 10% ethanol.(D) Amino acids 12 and 13 of Hax2 repeat units and target DNA sequenceof Hax2.(E) The promoter of PAP1 from A. thaliana Col-0 contains an imperfectHax2-box. Mismatches to the predicted Hax2-box are coloured in red. Aputative TATA-box, the natural transcription start site (+1), and thefirst codon of the PAP1 coding sequence are indicated.

FIG. 15. Table I. Predicted DNA target sequences of TAL effectors

The table shows repeat sequences of TAL effectors and the predicted DNAtarget sequences used from amino acids 12 and 13 of the repeat units.

The annotations show:

(A) Xcv, Xanthomonas campestris pv. vesicatoria; Xg, Xanthomonasgardneri; Xca, Xanthomonas campestris pv. armoraciae; Xoo, Xanthomonasoryzae pv. oryzae; Xac, Xanthomonas axonopodis pv. citri; Xau,Xanthomonas citri pv. aurantifolii; Xcm, Xanthomonas campestris pv.malvacearum; Xam, Xanthomonas axonopodis pv. manihotis; Xoc, Xanthomonasoryzae pv. oryzicola(B) A star (*) indicates a deletion of amino acid 13(C) Target DNA specificity deduced from amino acids 12 and 13 of therepeat units. A thymidine nucleotide is added at the 5′ end due to thespecificity of the putative repeat 0. The sequence of the upper (sense)strand of the double stranded DNA is given in ambiguous code (R=A/G;N=A/C/G/T; •=unknown specificity)

FIG. 16. Protein sequences of AvrBs3, Hax2, Hax3, Hax4

For each of the protein sequences, the N-terminus, C-terminus as well asthe single repeat sequences are shown.

FIG. 17. The effector ARTBs4 induces expression of the minimal Bs4promoter

(A) Amino acids 12 and 13 of the Hax4 repeat units and predicted targetDNA specificity (Hax4 box). The Hax4(mut) box contains four base pairexchanges in comparison to the Hax4 box.(B) Amino acids 12 and 13 of the artificial effector ARTBs4 repeat unitsand predicted target DNA specificity (ARTBs4 box).(C) The Hax4 box was cloned in front of the minimal Bs4 promoter into aGUS reporter vector. The ARTBs4 box is naturally present in the minimalBs4 promoter.(D) Specific inducibility of the Hax4 and ARTBs4 boxes by Hax4 andARTBs4, respectively. GUS reporter constructs were codelivered viaAgrobacterium tumefaciens into N. benthamiana with 35S-driven hax4 (greybars), ARTBs4 (white bars) and empty T-DNA (ev, black bars),respectively (error bars indicate SD). 4-MU, 4-methyl-umbelliferone.35S::uidA (GUS, grey bar) served as control. Leaf disks were stainedwith X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide).

FIG. 18. Diagram for “Golden gate” cloning of repeat domains andeffectors

(A) Building blocks consisting of individual repeat units (or otherprotein domains) are subcloned with flanking type II restriction enzymetarget sites (e.g. BsaI) that generate specific overhangs. Matchingoverhangs are indicated with identical letters (A to O). Differentrepeat types are cloned as building blocks for each position (e.g.repeat 1, repeat 2, etc.). The repeat specificities are: NI=A, HD=C,NG=T, NN=G or A.(B) The building blocks are assembled into a target vector by ligationof matching overhangs using “Golden gate” cloning(restriction-ligation). In general, the resulting assembly product doesnot contain any of the target sites used for cloning.

FIG. 19. Alternative method for generation of designer effectors viaGolden Gate cloning

FIGS. 19 A-D depict various vectors described in the methods disclosedin Example 3 below as well as provide a schematic of the method.

FIG. 20. Experiments to analyze novel repeat specificities

Artificial TALs were assembled with the first six repeats of the TALHax3. Repeat 7 to 11.5 were assembled using one repeat type with unknownspecificity. Four possible target DNA boxes were used containing six A,C, G, or T, respectively. Similarly, artificial TALs and reporter wereconstructed with 2, 3, or 4 repeats to test. The target DNA boxes wereinserted into the Bs4 minimal promoter upstream of a promoterless uidAreporter gene.

FIG. 21. TAL repeat specificities

Agrobacterium-mediated expression of artificial TALs and correspondingreporter constructs in Nicotiana benthamiana. Leaf disks were sampledtwo days post transformation, stained for GUS reporter activity anddestained with ethanol. A blue colour indicates expression of thereporter construct and therefore, an activity of the TAL. Empty vector(ev) and constitutively expressed GUS were used as negative control,respectively. Novel repeat specificities are colored in red. Repeattypes with strong DNA recognition properties are: NH, NP, NT, and HN.Repeat types with weak DNA recognition properties are: NG, N*, NK, SH,SN, IS.

FIG. 22. Quantitative analysis of known repeat specificities.

Artificial TALs were assembled with the first six repeats of the TALHax3. Repeat 7 to 11.5 were assembled using one repeat type. Fourpossible target DNA boxes were used containing six A, C, G, or T,respectively upstream of the Bs4 minimal promoter and a promoterlessuidA reporter gene. The data show that repeat type NN has much strongerDNA-recognition properties than the other repeat types. Repeat type NIis very weak and does not show a preference in this setup. Repeat typeNS was shown to recognice all four DNA bases, before, but does show apreference for A and G, here. EV: empty vector control.

FIG. 23. Quantitative analysis of novel repeats with multiplespecificities

Quantitative analysis of novel repeats with multiple specificities.Artificial TALs were assembled with the first six repeats of the TALHax3. Repeat 7 to 11.5 were assembled using one repeat type. Fourpossible target DNA boxes were used containing six A, C, G, or T,respectively upstream of the Bs4 minimal promoter and a promoterlessuidA reporter gene (see, FIG. 20).

FIG. 24. Quantitative analysis of novel repeats with only onespecificity

Artificial TALs were assembled with the first six repeats of the TALHax3. Repeat 7 to 11.5 were assembled using one repeat type. Fourpossible target DNA boxes were used containing six A, C, G, or T,respectively upstream of the Bs4 minimal promoter and a promoterlessuidA reporter gene. The data show that repeat type NH is much strongerthan repeat type NK, but also recognizes only one specific base (G).

FIG. 25. Quantitative analysis of novel repeats with novel specificities

Artificial TALs were assembled with the first six repeats of the TALHax3. Repeat 7 to 11.5 were assembled using one repeat type. Fourpossible target DNA boxes were used containing six A, C, G, or T,respectively upstream of the Bs4 minimal promoter and a promoterlessuidA reporter gene. These repeat types show only very low activity inthe reporter assay, likely due to their weak DNA interaction potential.

FIG. 26. Experimental setup to study specificity of repeat types withlow DNA recognition potential

The artificial effectors were assembled to contain 6, 4, 3, or 2repeats, respectively, with unknown specificity (designated XX) inaddition to Hax3 repeats. Target boxes in the reporter constructscontain A, C, G, or T, respectively, at positions corresponding to the“XX” repeats. The rest of the target DNA boxes is equivalent to the Hax3box.

FIG. 27A-C. Experimental setup to study specificity of repeat types withlow DNA recognition potential

The artificial effectors were assembled to contain 4, 3, or 2 repeats,respectively, as “test repeats” with unknown specificity (designated X)in addition to Hax3 repeats (see, FIG. 26 for details). Target boxes inthe reporter constructs contain A, C, G, or T, respectively, atpositions corresponding to the test repeats. The rest of the target DNAboxes is equivalent to the Hax3 box. Although TALs with four or morecombined N* repeats do not show a specificity, a combination of three ortwo N* repeats indicates a specificity for T, or T and C, respectively.N* and NI are obviously repeat types with weak DNA recognitionproperties. FIG. 27A: HD; FIG. 27B: N*; and FIG. 27C: NI.

SEQUENCE LISTING

The nucleotide and amino acid sequences listed in the accompanyingfigures and the sequence listing are shown using standard letterabbreviations for nucleotide bases, and one-letter code for amino acids.The nucleotide sequences follow the standard convention of beginning atthe 5′ end of the sequence and proceeding forward (i.e., from left toright in each line) to the 3′ end. Only one strand of each nucleic acidsequence is shown, but the complementary strand is understood to beincluded by any reference to the displayed strand. The amino acidsequences follow the standard convention of beginning at the aminoterminus of the sequence and proceeding forward (i.e., from left toright in each line) to the carboxy terminus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

A number of terms that are used throughout this disclosure are definedhereinbelow.

The term “repeat domain” is used to describe the DNA recognition domainfrom a TAL effector, or artificial version thereof that is made usingthe methods disclosed, consisting of modular repeat units that whenpresent in a polypeptide confer target DNA specificity. A repeat domaincomprised of repeat units can be added to any polypeptide in which DNAsequence targeting is desired and are not limited to use in TALeffectors.

The term “repeat unit” is used to describe the modular portion of arepeat domain from a TAL effector, or an artificial version thereof,that contains one amino acid or two adjacent amino acids that determinerecognition of a base pair in a target DNA sequence. Repeat units takentogether recognize a defined target DNA sequence and constitute a repeatdomain. Repeat units can be added to any polypeptide in which DNAsequence targeting is desired and are not limited to use in TALeffectors.

The term “recognition code” is used to describe the relationship betweenthe amino acids in positions 12 and 13 of a repeat unit and thecorresponding DNA base pair in a target DNA sequence that such aminoacids confer recognition of, as follows: HD for recognition of C/G; NIfor recognition of A/T; NG for recognition of T/A; NS for recognition ofC/G or A/T or T/A or G/C; NN for recognition of G/C or A/T; IG forrecognition of T/A; N for recognition of C/G or T/A; HG for recognitionof C/G or T/A; H for recognition of T/A; NK for recognition of G/C; NHfor recognition of G/C; NP for recognition of A/T, C/G, or T/A; NT forrecognition of A/T or G/C; NH for recognition of A/T or G/C; SH forrecognition of G/C; SN for recognition of G/C; and IS for recognition ofA/T. Additional specificities for the amino acids in positions inpositions 12 and 13 of a repeat unit and the corresponding DNA base pairin a target DNA sequence have been reported: HA for recognition of C/G;ND for recognition of C/G; HI for recognition of C/G; HN for recognitionof G/C; and NA for recognition of G/C (Moscou & Bogdanove (2009) Science326:1501).

As used herein, “effector” (or “effector protein” or “effectorpolypeptide”) refers to constructs or their encoded polypeptide productsin which said polypeptide is able to recognize a target DNA sequence.The effector protein includes a repeat domain comprised of 1.5 or morerepeat units and also may include one or more functional domains such asa regulatory domain. In preferred embodiments of the invention, the“effector” is additionally capable of exerting an effect, such asregulation of gene expression. Although the present invention is notdependent on a particularly biological mechanism, it is believe that theproteins or polypeptides of the invention that recognize a target DNAsequence bind to the target DNA sequence.

The term “naturally occurring” is used to describe an object that can befound in nature as distinct from being produced by man. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as-present in a wild-type individual, such as would be typicalfor the species.

The terms “modulating expression” “inhibiting expression” and“activating expression” of a gene refer to the ability of a polypeptideof the present invention to activate or inhibit transcription of a gene.Activation includes prevention of subsequent transcriptional inhibition(i.e., prevention of repression of gene expression) and inhibitionincludes prevention of subsequent transcriptional activation (i.e.,prevention of gene activation). Modulation can be assayed by determiningany parameter that is indirectly or directly affected by the expressionof the target gene. Such parameters include, e.g., changes in RNA orprotein levels, changes in protein activity, changes in product levels,changes in downstream gene expression, changes in reporter genetranscription (luciferase, CAT, beta-galactosidase, GFP (see, e.g.,Mistili & Spector (1997) Nature Biotechnology 15:961-964); changes insignal transduction, phosphorylation and dephosphorylation,receptor-ligand interactions, second messenger concentrations (e.g.,cGMP, cAMP, IP3, and Ca2+), cell growth, neovascularization, in vitro,in vivo, and ex vivo. Such functional effects can be measured by anymeans known to those skilled in the art, e.g., measurement of RNA orprotein levels, measurement of RNA stability, identification ofdownstream or reporter gene expression, e.g., via chemiluminescence,fluorescence, calorimetric reactions, antibody binding, induciblemarkers, ligand binding assays; changes in intracellular secondmessengers such as cGMP and inositol triphosphate (IP3); changes inintracellular calcium levels; cytokine release, and the like.

A “regulatory domain” refers to a protein or a protein subsequence thathas transcriptional modulation activity. Typically, a regulatory domainis covalently or non-covalently linked to a polypeptide of the presentinvention to modulate transcription. Alternatively, a polypeptide of thepresent invention can act alone, without a regulatory domain, or withmultiple regulatory domains to modulate transcription. Transcriptionfactor polypeptides from which one can obtain a regulatory domaininclude those that are involved in regulated and basal transcription.Such polypeptides include transcription factors, their effector domains,coactivators, silencers, nuclear hormone receptors (see, e.g., Goodrichet al. (1996) Cell 84:825 30 for a review of proteins and nucleic acidelements involved in transcription; transcription factors in general arereviewed in Barnes & Adcock (1995) Clin. Exp. Allergy 25 Suppl. 2:46 9and Roeder (1996) Methods Enzymol. 273:165 71). Databases dedicated totranscription factors are known (see, e.g., Science (1995) 269:630).Nuclear hormone receptor transcription factors are described in, forexample, Rosen et al. (1995) J. Med. Chem. 38:4855 74. The C/EBP familyof transcription factors are reviewed in Wedel et al. (1995)Immunobiology 193:171 85. Coactivators and co-repressors that mediatetranscription regulation by nuclear hormone receptors are reviewed in,for example, Meier (1996) Eur. J. Endocrinol. 134(2):158 9; Kaiser etal. (1996) Trends Biochem. Sci. 21:342 5; and Utley et al. (1998) Nature394:498 502). GATA transcription factors, which are involved inregulation of hematopoiesis, are described in, for example, Simon (1995)Nat. Genet. 11:9 11; Weiss et al. (1995) Exp. Hematol. 23:99-107. TATAbox binding protein (TBP) and its associated TAF polypeptides (whichinclude TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250) are describedin Goodrich & Tjian (1994) Curr. Opin. Cell Biol. 6:403 9 and Hurley(1996) Curr. Opin. Struct. Biol. 6:69 75. The STAT family oftranscription factors are reviewed in, for example, Barahmand-Pour etal. (1996) Curr. Top. Microbiol. Immunol. 211:121 8. Transcriptionfactors involved in disease are reviewed in Aso et al. (1996) J. Clin.Invest. 97:1561 9. Kinases, phosphatases, and other proteins that modifypolypeptides involved in gene regulation are also useful as regulatorydomains for polypeptides of the present invention. Such modifiers areoften involved in switching on or off transcription mediated by, forexample, hormones. Kinases involved in transcription regulation arereviewed in Davis (1995) Mol. Reprod. Dev. 42:459 67, Jackson et al.(1993) Adv. Second Messenger Phosphoprotein Res. 28:279 86, and Boulikas(1995) Crit. Rev. Eukaryot. Gene Expr. 5:1 77, while phosphatases arereviewed in, for example, Schonthal & Semin (1995) Cancer Biol. 6:23948. Nuclear tyrosine kinases are described in Wang (1994) TrendsBiochem. Sci. 19:373 6. Useful domains can also be obtained from thegene products of oncogenes (e.g., myc, jun, fos, myb, max, mad, rel,ets, bcl, myb, mos family members) and their associated factors andmodifiers. Oncogenes are described in, for example, Cooper, Oncogenes,2nd ed., The Jones and Bartlett Series in Biology, Boston, Mass., Jonesand Bartlett Publishers, 1995. The ets transcription factors arereviewed in Waslylk et al. (1993) Eur. J. Biochem. 211:7 18 and Crepieuxet al. (1994) Crit. Rev. Oncog. 5:615 38. Myc oncogenes are reviewed in,for example, Ryan et al. (1996) Biochem. J. 314:713 21. The jun and fostranscription factors are described in, for example, The Fos and JunFamilies of Transcription Factors, Angel & Herrlich, eds. (1994). Themax oncogene is reviewed in Hurlin et al. Cold Spring Harb. Symp. Quant.Biol. 59:109 16. The myb gene family is reviewed in Kanei-Ishii et al.(1996) Curr. Top. Microbiol. Immunol. 211:89 98. The mos family isreviewed in Yew et al. (1993) Curr. Opin. Genet. Dev. 3:19 25.Polypeptides of the present invention can include regulatory domainsobtained from DNA repair enzymes and their associated factors andmodifiers. DNA repair systems are reviewed in, for example, Vos (1992)Curr. Opin. Cell Biol. 4:385 95; Sancar (1995) Ann. Rev. Genet. 29:69105; Lehmann (1995) Genet. Eng. 17:1 19; and Wood (1996) Ann. Rev.Biochem. 65:135 67. DNA rearrangement enzymes and their associatedfactors and modifiers can also be used as regulatory domains (see, e.g.,Gangloff et al. (1994) Experientia 50:261 9; Sadowski (1993) FASEB J.7:760 7).

Similarly, regulatory domains can be derived from DNA modifying enzymes(e.g., DNA methyltransferases, topoisomerases, helicases, ligases,kinases, phosphatases, polymerases) and their associated factors andmodifiers. Helicases are reviewed in Matson et al. (1994) Bioessays16:13 22, and methyltransferases are described in Cheng (1995) Curr.Opin. Struct. Biol. 5:4 10. Chromatin associated proteins and theirmodifiers (e.g., kinases, acetylases and deacetylases), such as histonedeacetylase (Wolffe Science 272:371 2 (1996)) are also useful as domainsfor addition to the effector of choice. In one preferred embodiment, theregulatory domain is a DNA methyl transferase that acts as atranscriptional repressor (see, e.g., Van den Wyngaert et al. FEBS Lett.426:283 289 (1998); Flynn et al. J. Mol. Biol. 279:101 116 (1998); Okanoet al. Nucleic Acids Res. 26:2536 2540 (1998); and Zardo & Caiafa, J.Biol. Chem. 273:16517 16520 (1998)). In another preferred embodiment,endonucleases such as FokI are used as transcriptional repressors, whichact via gene cleavage (see, e.g., WO95/09233; and PCT/US94/01201).Factors that control chromatin and DNA structure, movement andlocalization and their associated factors and modifiers; factors derivedfrom microbes (e.g., prokaryotes, eukaryotes and virus) and factors thatassociate with or modify them can also be used to obtain chimericproteins. In one embodiment, recombinases and integrases are used asregulatory domains. In one embodiment, histone acetyltransferase is usedas a transcriptional activator (see, e.g., Jin & Scotto (1998) Mol.Cell. Biol. 18:4377 4384; Wolffe (1996) Science 272:371 372; Taunton etal. Science 272:408 411 (1996); and Hassig et al. PNAS 95:3519 3524(1998)). In another embodiment, histone deacetylase is used as atranscriptional repressor (see, e.g., Jin & Scotto (1998) Mol. Cell.Biol. 18:4377 4384; Syntichaki & Thireos (1998) J. Biol. Chem. 273:2441424419; Sakaguchi et al. (1998) Genes Dev. 12:2831 2841; and Martinez etal. (1998) J. Biol. Chem. 273:23781 23785).

As used herein, “gene” refers to a nucleic acid molecule or portionthereof which comprises a coding sequence, optionally containingintrons, and control regions which regulate the expression of the codingsequence and the transcription of untranslated portions of thetranscript.

Thus, the term “gene” includes, besides coding sequence, regulatorysequence such as the promoter, enhancer, 5′ untranslated regions, 3′untranslated region, termination signals, poly adenylation region andthe like. Regulatory sequence of a gene may be located proximal to,within, or distal to the coding region.

As used herein, “target gene” refers to a gene whose expression is to bemodulated by a polypeptide of the present invention.

As used herein, “plant” refers to any of various photosynthetic,eucaryotic multi-cellular organisms of the kingdom Plantae,characteristically producing embryos, containing chloroplasts, havingcellulose cell walls and lacking locomotion. As used herein, “plant”includes any plant or part of a plant at any stage of development,including seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,microspores, and progeny thereof. Also included are cuttings, and cellor tissue cultures. As used in conjunction with the present invention,the term “plant tissue” includes, but is not limited to, whole plants,plant cells, plant organs, e.g., leafs, stems, roots, meristems, plantseeds, protoplasts, callus, cell cultures, and any groups of plant cellsorganized into structural and/or functional units.

As used herein, “modulate the expression of a target gene in plantcells” refers to increasing (activation) or decreasing (repression) theexpression of the target gene in plant cells with a polypeptide of thepresent invention, alone or in combination with other transcriptionand/or translational regulatory factors, or nucleic acids encoding suchpolypeptide, in plant cells.

As used herein, a “target DNA sequence” refers to a portion ofdouble-stranded DNA to which recognition by a protein is desired. In oneembodiment, a “target DNA sequence” is all or part of a transcriptionalcontrol element for a gene for which a desired phenotypic result can beattained by altering the degree of its expression. A transcriptionalcontrol element includes positive and negative control elements such asa promoter, an enhancer, other response elements, e.g., steroid responseelement, heat shock response element, metal response element, arepressor binding site, operator, and/or a silencer. The transcriptionalcontrol element can be viral, eukaryotic, or prokaryotic. A “target DNAsequence” also includes a downstream or an upstream sequence which canbind a protein and thereby modulate, typically prevent, transcription.

The use of the term “DNA” or “DNA sequence” herein is not intended tolimit the present invention to polynucleotide molecules comprising DNA.Those of ordinary skill in the art will recognize that the methods andcompositions of the invention encompass polynucleotide moleculescomprised of deoxyribonucleotides (i.e., DNA), ribonucleotides (i.e.,RNA) or combinations of ribonucleotides and deoxyribonucleotides. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues including, but not limitedto, nucleotide analogs or modified backbone residues or linkages, whichare synthetic, naturally occurring, and non-naturally occurring, whichhave similar binding properties as the reference nucleic acid, and whichare metabolized in a manner similar to the reference nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Thepolynucleotide molecules of the invention also encompass all forms ofpolynucleotide molecules including, but not limited to, single-strandedforms, double-stranded forms, hairpins, stem-and-loop structures, andthe like. Furthermore, it is understood by those of ordinary skill inthe art that the DNA sequences disclosed herein also encompasses thecomplement of that exemplified nucleotide sequence.

As used herein, “specifically binds to a target DNA sequence” means thatthe binding affinity of a polypeptide of the present invention to aspecified target DNA sequence is statistically higher than the bindingaffinity of the same polypeptide to a generally comparable, butnon-target DNA sequence. It also refers to binding of a repeat domain ofthe present invention to a specified target DNA sequence to a detectablygreater degree, e.g., at least 1.5-fold over background, than itsbinding to non-target DNA sequences and to the substantial exclusion ofnon-target DNA sequences. A polypeptide of the present invention's Kd toeach DNA sequence can be compared to assess the binding specificity ofthe polypeptide to a particular target DNA sequence.

As used herein, a “target DNA sequence within a target gene” refers to afunctional relationship between the target DNA sequence and the targetgene in that recognition of a polypeptide of the present invention tothe target DNA sequence will modulate the expression of the target gene.The target DNA sequence can be physically located anywhere inside theboundaries of the target gene, e.g., 5′ ends, coding region, 3′ ends,upstream and downstream regions outside of cDNA encoded region, orinside enhancer or other regulatory region, and can be proximal ordistal to the target gene.

As used herein, “endogenous” refers to nucleic acid or protein sequencenaturally associated with a target gene or a host cell into which it isintroduced.

As used herein, “exogenous” refers to nucleic acid or protein sequencenot naturally associated with a target gene or a host cell into which itis introduced, including non-naturally occurring multiple copies of anaturally occurring nucleic acid, e.g., DNA sequence, or naturallyoccurring nucleic acid sequence located in a non-naturally occurringgenome location.

As used herein, “genetically modified plant (or transgenic plant)”refers to a plant which comprises within its genome an exogenouspolynucleotide. Generally, and preferably, the exogenous polynucleotideis stably integrated within the genome such that the polynucleotide ispassed on to successive generations. The exogenous polynucleotide may beintegrated into the genome alone or as part of a recombinant expressioncassette. “Transgenic” is used herein to include any cell, cell line,callus, tissue, plant part or plant, the genotype of which has beenaltered by the presence of exogenous nucleic acid including thosetransgenics initially so altered as well as those created by sexualcrosses or asexual propagation from the initial transgenic. The term“transgenic” as used herein does not encompass the alteration of thegenome (chromosomal or extra-chromosomal) by conventional plant breedingmethods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

As used herein, “minimal promoter” or substantially similar term refersto a promoter element, particularly a TATA element, that is inactive orthat has greatly reduced promoter activity in the absence of upstreamactivation. In the presence of a suitable transcription factor, theminimal promoter functions to permit transcription.

As used herein, “repressor protein” or “repressor” refers to a proteinthat binds to operator of DNA or to RNA to prevent transcription ortranslation, respectively.

As used herein, “repression” refers to inhibition of transcription ortranslation by binding of repressor protein to specific site on DNA ormRNA. Preferably, repression includes a significant change intranscription or translation level of at least 1.5 fold, more preferablyat least two fold, and even more preferably at least five fold.

As used herein, “activator protein” or “activator” refers to a proteinthat binds to operator of DNA or to RNA to enhance transcription ortranslation, respectively.

As used herein, “activation” refers to enhancement of transcription ortranslation by binding of activator protein to specific site on DNA ormRNA. Preferably, activation includes a significant change intranscription or translation level of at least 1.5 fold, more preferablyat least two fold, and even more preferably at least five fold.

As used herein, “derivative” or “analog” of a molecule refers to aportion derived from or a modified version of the molecule.

As used herein, a “repeat unit derived from a transcriptionactivator-like (TAL) effector” refers to a repeat unit from a TALeffector or a modified or artificial version of one or more TALeffectors that is produced by any of the methods disclosed herein.

In the following, the invention is specifically described with respectto the transcription activator-like (TAL) effector family which aretranslocated via the type III secretion system into plant cells. Thetype member of this effector family is AvrBs3. Hence, the TAL effectorfamily is also named AvrBs3-like family of proteins. Both expressionsare used synonymously and can be interchanged. Non-limiting examples ofthe AvrBs3-like family are as follows: AvrBs4 and the members of the Haxsub-family Hax2, Hax3, and Hax4 as well as Brg11. AvrBs3 and the othermembers of its family are characterized by their binding capability tospecific DNA sequences in promoter regions of target genes and inductionof expression of these genes. They have conserved structural featuresthat enable them to act as transcriptional activators of plant genes.AvrBs3-like family and homologous effectors typically have in theirC-terminal region nuclear localisation sequences (NLS) and atranscriptional activation domain (AD). The central region containsrepeat units of typically 34 or 35 amino acids. The repeat units arenearly identical, but variable at certain positions and it has now beenfound how these positions determine the nucleotide sequence bindingspecificity of the proteins.

It was shown for AvrBs3 that the repeat units are responsible forbinding to DNA. The DNA-binding specificity of AvrBs3 and probably othermembers of the AvrBs3-family seems to be mediated by the central repeatdomain of the proteins. This repeat domain consists in AvrBs3 of 17.5repeat units and in homologous proteins is comprised of 1.5 to 33.5repeat units which are typically 34 amino acids each. Other repeat unitlengths are also known (e.g. 30, 33, 35, 39, 40, 42 amino acids). Thelast repeat in the repeat domain is usually only a half repeat of 19 or20 amino acids length. The individual repeat units are generally notidentical. They vary at certain variable amino acid positions, amongthese positions 12 and 13 are hypervariable while positions 4, 11, 24,and 32 vary with high frequency but at a lower frequency than 12 and 13(variations at other positions occur also, but at lower frequency). Thecomparison of different AvrBs3-like proteins from Xanthomonas reveals 80to 97% overall sequence identity with most differences confined to therepeat domain. For example, AvrBs3 and the AvrBs3-like family memberAvrBs4 differ exclusively in their repeat domain region, with theexception of a four amino acid deletion in the C-terminus of AvrBs4 withrespect to AvrBs3.

In FIG. 16, the amino acid sequences of AvrBs3 as well as the amino acidsequences of the members of the Hax-sub family are shown. Of particularimportance for the present invention is the repeat units, which areidentical except for the hypervariable amino acids at positions 12 and13 and the variable amino acids at positions 4 and 24. Hence, eachrepeat unit of these proteins is given separately.

As stated above, it has already been described that the repeat unitswithin the repeat domains determine recognition or binding capabilityand specificity of type III effector proteins of AvrBs3-family. However,the principle underlying was not known until the present invention.

The inventors have discovered that one repeat unit within a repeatdomain is responsible for the recognition of one specific DNA base pairin a target DNA sequence. This finding is, however, only one element ofthe invention. The inventors additionally discovered that ahypervariable region within each repeat unit of a repeat domain isresponsible for recognition of one specific DNA base pair in a targetDNA sequence. Within a repeat unit, the hypervariable region(corresponds to amino acid positions 12 and 13) are typicallyresponsible for this recognition specificity. Hence each variation inthese amino acids reflects a corresponding variation in target DNArecognition and preferably also recognition capacity.

As used herein, “hypervariable region” is intended to mean positions 12and 13 or equivalent position in a repeat unit of the present invention.It is recognized that positions 12 and 13 of the invention correspond topositions 12 and 13 in the full-length repeat units of AvrBs3 and otherTAL effectors as disclosed herein. It is further recognized that by“equivalent positions” is intended positions that corresponds topositions 12 and 13, respectively, in a repeat unit of the present. Onecan readily determine such equivalent positions by aligning any repeatunit with a full-length repeat unit of AvrBs3.

It has, therefore, been shown for the first time that one repeat unit ina repeat domain of a DNA-binding protein recognizes one base pair in thetarget DNA, and that one amino acid or two adjacent amino acid residuesin a repeat unit, typically within the hypervariable regions of a repeatunit, determine which base pair in the target DNA is recognized. Basedon this finding, a person skilled in the art would be able tospecifically target base pairs in a target DNA sequence of interest bymodifying a polypeptide within its repeat units of the repeat domain tospecifically target base pairs in the desired target DNA sequence. Basedon this finding, the inventors have identified a recognition code forDNA-target specificities of different repeat types and were able topredict target DNA sequences of several TAL effectors which could beconfirmed experimentally. This will additionally facilitate theidentification of host genes that are regulated by TAL effectors. Thelinear array of repeat units which recognizes a linear sequence of basesin the target DNA is a novel DNA-protein interaction. The modulararchitecture of the repeat domain and the recognition code identified bythe inventors for targeting DNA with high specificity allows theefficient design of specific DNA-binding domains for use in a variety oftechnological fields.

In one embodiment of the present invention, the repeat domains areincluded in a transcription factor, for instance in transcriptionfactors active in plants, particularly preferred in type III effectorproteins, e.g. in effectors of the AvrBs3-like family. However, afterhaving uncovered the correlation between the repeat units in a repeatdomain on the one hand and the base sequence in the target DNA on theother hand, the modular architecture of the repeat domain can be used inany protein which shall be used for targeting specific target DNAsequences. By introducing repeat domains comprising repeat units into apolypeptide wherein the repeat units are modified in order to compriseone hypervariable region per repeat unit and wherein the hypervariableregion determines recognition of a base pair in a target DNA sequence,the recognition of a large variety of proteins to pre-determined targetDNA sequences will be available.

As one repeat unit within a repeat domain has been found to beresponsible for the specific recognition of one base pair in a DNA,several repeat units can be combined with each other wherein each repeatunit includes a hypervariable region that is responsible for therecognition of each repeat unit to a particular base pair in a targetDNA sequence.

Techniques to specifically modify DNA sequences in order to obtain aspecified codon for a specific amino acid are known in the art.

Methods for mutagenesis and polynucleotide alterations have been widelydescribed. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. All these publications are herein incorporatedby reference.

The following examples provide methods for constructing new repeat unitsand testing the specific binding activities of artificially constructedrepeat units specifically recognizing base pairs in a target DNAsequence.

The number of repeat units to be used in a repeat domain can beascertained by one skilled in the art by routine experimentation.Generally, at least 1.5 repeat units are considered as a minimum,although typically at least about 8 repeat units will be used. Therepeat units do not have to be complete repeat units, as repeat units ofhalf the size can be used. Moreover, the methods and polypeptidesdisclosed herein do depend on repeat domains with a particular number ofrepeat units. Thus, a polypeptide of the invention can comprise, forexample, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23,23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30,30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37,37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44,44.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5 or more repeatunits. Typically, AvrBs3 contains 17.5 repeat units and inducesexpression of UPA (up-regulated by AvrBs3) genes. The number and orderof repeat units will determine the corresponding activity and DNArecognition specificity. As further examples, the AvrBs3 family membersHax2 includes 21.5 repeat units, Hax3 11.5 repeat units and Hax4 14.5repeat units. Preferably, a polypeptide of the invention comprises about8 and to about 39 repeat units. More preferably, a polypeptide of theinvention comprises about 11.5 to about 33.5 repeat units.

A typical consensus sequence of a repeat with 34 amino acids (inone-letter code) is shown below:

LTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG

A further consensus sequence for a repeat unit with 35 amino acids (inone-letter code) is as follows:

LTPEQVVAIASNGGGKQALETVQRLLPVLCQAPHD

The repeat units which can be used in one embodiment of the inventionhave an identity with the consensus sequences described above of atleast 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%. In preferredembodiments, the repeat sequences of AvrBs3, Hax2, Hax3 and Hax4 andfurther members of the AvrBs3-family are used. The repeat unit sequencesof these members are indicated in FIG. 16. These repeat unit sequencescan be modified by exchanging one or more of the amino acids. Themodified repeat unit sequences have an identity with the original repeatsequence of the original member of the AvrBs3-family sequence of atleast 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%. In preferredembodiments, the amino acids in positions

12 and 13 are altered. In still further embodiments, amino acids inpositions 4, 11, 24, and 32 are altered. Preferably, the number of aminoacids per repeat are in a range between 20-45 amino acids, furthermore32-40 amino acids, still further 32-39 amino acid, and furtheroptionally 32, 34, 35 or 39 amino acids per repeat unit.

Specifically, the hypervariable region in a repeat unit determines thespecific recognition of one base pair in a target DNA sequence. Morespecifically, the inventors have found the following correlation ofrecognition specificity between amino acids found at positions 12 and 13in a repeat unit and base pairs in the target DNA sequence:

-   -   HD for recognition of C/G    -   NI for recognition of A/T    -   NG for recognition of T/A    -   NS for recognition of C/G or A/T or T/A or G/C    -   NN for recognition of G/C or A/T    -   IG for recognition of T/A    -   N for recognition of C/G or T/A    -   HG for recognition of T/A    -   H for recognition of T/A    -   NK for recognition of G/C    -   NH for recognition of G/C    -   NP for recognition of A/T or C/G or T/A    -   NT for recognition of A/T or G/C    -   HN for recognition of A/T or G/C    -   SH for recognition of G/C    -   SN for recognition of G/C and    -   IS for recognition of A/T.

It has to be noted that the amino acids are represented in the singleletter code. The nucleotides are given as base pairs, wherein the firstbase is located in the upper strand and the second base in the lowerstrand; for example C/G means that C is located in the upper strand, Gin the lower strand.

The methods of the present invention can further comprise making arepeat unit in which one or more of the hypervariable regions isselected from the following group in order to determine recognition ofone of the following base pairs: HA for recognition of C/G; ND forrecognition of C/G; HI for recognition of C/G; HN for recognition ofG/C; and NA for recognition of G/C.

With respect to the single amino acids N and H, respectively, amino acid13 of AvrBs3 appears to be missing from the repeat unit when compared bymultiple amino acid sequence alignments with the other repeat units.

In one embodiment of the invention, the N-terminal domain of AvrBs3-likeproteins confers recognition specificity for a T, 5′ of the recognitionspecificity of said repeat.

In a particularly preferred embodiment of the invention, repeat units ofthe protein family AvrBs3 are used. Examples for the members of thisprotein family have been specified above. Particularly, the members ofthe protein family have an amino acid homology of at least 95%, at least90%, at least 80%, at least 85%, at least 70%, at least 75%, at least60%, at least 50%, at least 40% or at least 35% to the amino acidsequence of AvrBs3, particularly to the amino acid sequence of therepeat unit of AvrBs3. Having this in mind, the hypervariable region ina repeat unit can be deduced by an amino acid comparison between themembers of the AvrBs3 family. In particularly preferred embodiments, theamino acids are in positions 12 and 13 of a repeat unit of AvrBs3.However, variable regions may also be located in different amino acidpositions. Examples for variable positions are amino acids numbers 4,11, 24, and 32. In a further embodiment of the invention, the aminoacids responsible for the specific recognition of a base pair in a DNAsequence are located in positions which typically do not vary betweenthe members of the AvrBs3 family or in positions which are variable butnot hypervariable.

To summarize, the inventors have found that repeat units determine therecognition of one base pair on a DNA sequence and that thehypervariable region within a repeat unit determines the recognitionspecificity of the corresponding repeat unit. Hence, the sequence ofrepeat units correlates with a specific linear order of base pairs in atarget DNA sequence. The inventors have found this correlation withrespect to AvrBs3 and verified it with respect to a representativenumber of members of the AvrBs3-like family of proteins. With respect toAvrBs3-like family members, amino acid residues in positions 12 and 13in a repeat unit of 34 or other amino acids length correlate withdefined binding specificities of AvrBs3-like proteins. The discovery ofthis core principle provides a powerful tool to customize a polypeptidewith its cognate target DNA template for a variety of applicationsincluding, but not limited to, modulation of gene expression andtargeted genome engineering.

In the present invention, polypeptides can be designed which comprise arepeat domain with repeat units wherein in the repeat unitshypervariable regions are included which determine recognition of a basepair in a target DNA sequence. In one embodiment of the invention, eachrepeat unit includes a hypervariable region which determine recognitionof one base pair in a target DNA sequence. In a further embodiment, 1 or2 repeat units in a repeat domain are included which do not specificallyrecognize a base pair in a target DNA sequence. Considering therecognition code found by the inventors, a modular arrangement of repeatunits is feasible wherein each repeat unit is responsible for thespecific recognition of one base pair in a target DNA sequence.Consequently, a sequence of repeat units corresponds to a sequence ofbase pairs in a target DNA sequence so that 1 repeat unit matches to onebase pair.

Provided that a target DNA sequence is known and to which recognition bya protein is desired, the person skilled in the art is able tospecifically construct a modular series of repeat units, includingspecific recognition amino acid sequences, and assemble these repeatunits into a polypeptide in the appropriate order to enable recognitionof and binding to the desired target DNA sequence. Any polypeptide canbe modified by being combined with a modular repeat unit DNA-bindingdomain of the present invention. Such examples include polypeptides thatare transcription activator and repressor proteins, resistance-mediatingproteins, nucleases, topoisomerases, ligases, integrases, recombinases,resolvases, methylases, acetylases, demethylases, deacetylases, and anyother polypeptide capable of modifying DNA, RNA, or proteins.

The modular repeat unit DNA-binding domain of the present invention canbe combined with cell compartment localisation signals such as nuclearlocalisation signals, to function at any other regulatory regions,including but not limited to, transcriptional regulatory regions andtranslational termination regions.

In a further embodiment of the invention, these modularly designedrepeat units are combined with an endoneclease domain capable ofcleaving DNA when brought into proximity with DNA as a result of bindingby the repeat domain. Such endonucleolytic breaks are known to stimulatethe rate of homologous recombination in eukaryotes, including fungi,plants, and animals. The ability to simulate homologous recombination ata specific site as a result of a site-specific endonucleolytic breakallows the recovery of transformed cells that have integrated a DNAsequence of interest at the specific site, at a much higher frequencythan is possible without having made the site-specific break. Inaddition, endonucleolytic breaks such as those caused by polypeptidesformed from a repeat domain and an endonuclease domain are sometimesrepaired by the cellular DNA metabolic machinery in a way that altersthe sequence at the site of the break, for instance by causing a shortinsertion or deletion at the site of the break compared to the unalteredsequence. These sequence alterations can cause inactivation of thefunction of a gene or protein, for instance by altering a protein-codingsequence to make a non-functional protein, modifying a splice site sothat a gene transcript is not properly cleaved, making a non-functionaltranscript, changing the promoter sequence of a gene so that it can nolonger by appropriately transcribed, etc.

Breaking DNA using site specific endonucleases can increase the rate ofhomologous recombination in the region of the breakage. In someembodiments, the Fok I (Flavobacterium okeanokoites) endonuclease may beutilized in an effector to induce DNA breaks. The Fok I endonucleasedomain functions independently of the DNA binding domain and cuts adouble stranded DNA typically as a dimer (Li et al. (1992) Proc. Natl.Acad. Sci. U.S.A 89 (10):4275-4279, and Kim et al. (1996) Proc. Natl.Acad. Sci. U.S.A 93 (3):1156-1160; the disclosures of which areincorporated herein by reference in their entireties). A single-chainFokI dimer has also been developed and could also be utilized (Mino etal. (2009) J. Biotechnol. 140:156-161). An effector could be constructedthat contains a repeat domain for recognition of a desired target DNAsequence as well as a FokI endonuclease domain to induce DNA breakage ator near the target DNA sequence similar to previous work done employingzinc finger nucleases (Townsend et al. (2009) Nature 459:442-445; Shuklaet al. (2009) Nature 459, 437-441, all of which are herein incorporatedby reference in their entireties). Utilization of such effectors couldenable the generation of targeted changes in genomes which includeadditions, deletions and other modifications, analogous to those usesreported for zinc finger nucleases as per Bibikova et al. (2003) Science300, 764; Urnov et al. (2005) Nature 435, 646; Wright et al. (2005) ThePlant Journal 44:693-705; and U.S. Pat. Nos. 7,163,824 and 7,001,768,all of which are herein incorporated by reference in their entireties.

The FokI endonuclease domain can be cloned by PCR from the genomic DNAof the marine bacteria Flavobacterium okeanokoites (ATCC) prepared bystandard methods. The sequence of the FokI endonuclease is available onPubmed (Acc. No. M28828 and Acc. No J04623, the disclosures of which areincorporated herein by reference in their entireties). The I-Sce Iendonuclease from the yeast Saccharomyces cerevisiae has been used toproduce DNA breaks that increase the rate of homologous recombination.I-Sce I is an endonuclease encoded by a mitochondrial intron which hasan 18 bp recognition sequence, and therefore a very low frequency ofrecognition sites within a given DNA, even within large genomes (Thierryet al. (1991) Nucleic Acids Res. 19 (1):189-190; the disclosure of whichis incorporated herein by reference in its entirety). The infrequency ofcleavage sites recognized by I-SceI makes it suitable to use forenhancing homologous recombination. Additional description regarding theuse of I-Sce Ito induce said DNA breaks can be found in U.S. Pat. Appl.20090305402, which is incorporated herein by reference in its entirety.

The recognition site for I-Sce I has been introduced into a range ofdifferent systems. Subsequent cutting of this site with I-Sce Iincreases homologous recombination at the position where the site hasbeen introduced. Enhanced frequencies of homologous recombination havebeen obtained with I-Sce I sites introduced into the extra-chromosomalDNA in Xenopus oocytes, the mouse genome, and the genomic DNA of thetobacco plant Nicotiana plumbaginifolia. See, for example, Segal et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92 (3):806-810; Choulika et al.(1995) Mol. Cell. Biol. 15 (4):1968-1973; and Puchta et al. (1993)Nucleic Acids Res. 21 (22):5034-5040; the disclosures of which areincorporated herein by reference in their entireties. It will beappreciated that any other endonuclease domain that works withheterologous DNA binding domains can be utilized in an effector and thatthe I-Sce I endonuclease is one such non-limiting example. Thelimitation of the use of endonucleases that have a DNA recognition andbinding domain such as I-Sce I is that the recognition site has to beintroduced by standard methods of homologous recombination at thedesired location prior to the use of said endonuclease to enhancehomologous recombination at that site, if such site is not alreadypresent in the desired location. Methods have been reported that enablethe design and synthesis of novel endonucleases, such as by modifyingknown endonucleases or making chimeric versions of one or more suchendonucleases, that recognize novel target DNA sequences, thus pavingthe way for generation of such engineered endonuclease domains to cleaveendogenous target DNA sequences of interest (Chevalier et al. (2002)Molecular Cell 10:895-905; WO2007/060495; WO2009/095793; Fajardo-Sanchezet al. (2008) Nucleic Acids Res. 36:2163-2173, both of which areincorporated by reference in their entireties). As such, it could beenvisioned that such endonuclease domains could be similarly engineeredso as to render the DNA-binding activity non-functional but leaving theDNA cleaving function active and to utilize said similarly engineeredendonuclease cleavage domain in an effector to induce DNA breaks similarto the use of FokI above. In such applications, target DNA sequencerecognition would preferably be provided by the repeat domain of theeffector but DNA cleavage would be accomplished by the engineeredendonuclease domain.

As mentioned above, an effector includes a repeat domain with specificrecognition for a desired specific target sequence. In preferredembodiments, the effector specifically binds to an endogenouschromosomal DNA sequence. The specific nucleic acid sequence or morepreferably specific endogenous chromosomal sequence can be any sequencein a nucleic acid region where it is desired to enhance homologousrecombination. For example, the nucleic acid region may be a regionwhich contains a gene in which it is desired to introduce a mutation,such as a point mutation or deletion, or a region into which it isdesired to introduce a gene conferring a desired phenotype.

Further embodiments relate to methods of generating a modified plant inwhich a desired addition has been introduced. The methods can includeobtaining a plant cell that includes an endogenous target DNA sequenceinto which it is desired to introduce a modification; generating adouble-stranded cut within the endogenous target DNA sequence with aneffector that includes a repeat domain that binds to an endogenoustarget DNA sequence and an endonuclease domain;

introducing an exogenous nucleic acid that includes a sequencehomologous to at least a portion of the endogenous target DNA into theplant cell under conditions which permit homologous recombination tooccur between the exogenous nucleic acid and the endogenous target DNAsequence; and generating a plant from the plant cell in which homologousrecombination has occurred. Other embodiments relate to geneticallymodified cells and plants made according to the method described aboveand herein. It should be noted that the target DNA sequence could beartificial or naturally occurring. It will be appreciated that suchmethods could be used in any organism (such non-limiting organisms toinclude animals, humans, fungi, oomycetes bacteria and viruses) usingtechniques and methods known in the art and utilized for such purposesin such organisms.

In a further embodiment of the invention, these modularly designedrepeat domains are combined with one or more domains responsible for themodulation or control of the expression of a gene, for instance of plantgenes, animal genes, fungal genes, oomycete genes, viral genes, or humangenes. Methods for modulating gene expression by generating DNA-bindingpolypeptides containing zinc finger domains is known in the art (U.S.Pat. Nos. 7,285,416, 7,521,241, 7,361,635, 7,273,923, 7,262,054,7,220,719, 7,070,934, 7,013,219, 6,979,539, 6,933,113, 6,824,978, eachof which is hereby herein incorporated by reference in its entirety).For instance, these effectors of the AvrBs3-like family are modified inorder to bind to specific target DNA sequences. Such polypeptides mightfor instance be transcription activators or repressor proteins oftranscription which are modified by the method of the present inventionto specifically bind to genetic control regions in a promoter of orother regulatory region for a gene of interest in order to activate,repress or otherwise modulate transcription of said gene.

In a still further embodiment of the invention, the target DNA sequencesare modified in order to be specifically recognized by a naturallyoccurring repeat domain or by a modified repeat domain. As one example,the target DNA sequences for members of the AvrBs3-like family can beinserted into promoters to generate novel controllable promoters thatcan be induced by the corresponding AvrBs3 effector. Secondary induciblesystems can be constructed using a trans-activator and a target gene,wherein the trans-activator is a polypeptide wherein said polypeptidecomprises at least a repeat domain comprising repeat units of thepresent invention that bind to said target gene and induce expression.The trans-activator and the target gene can be introduced into one cellline but may also be present in different cell lines and later beintrogressed. In a further embodiment, disease-resistant plants can beconstructed by inserting the target DNA sequence of a repeat domaincontaining polypeptide of the present invention in front of a gene whichafter expression leads to a defence reaction of the plant by activatinga resistance-mediating gene.

In a further embodiment, custom DNA-binding polypeptides can beconstructed by rearranging repeat unit types thus allowing thegeneration of repeat domains with novel target DNA binding specificity.Individual repeat units are nearly identical at the DNA level whichprecludes classical cloning strategies. The present invention provides aquick and inexpensive strategy to assemble custom polypeptides withrepeat domains of the present invention. To improve cloning versatilitysuch polypeptides, a two-step assembly method was designed. This methodwas used to assemble polypeptides with novel repeat types to study theirtarget DNA recognition and binding specificity.

Summarily, any DNA sequence can be modified to enable binding by arepeat domain containing polypeptide of the present invention byintroducing base pairs into any DNA region or specific regions of a geneor a genetic control element to specifically target a polypeptide havinga repeat domain comprised of repeat units that will bind said modifiedDNA sequence in order to facilitate specific recognition and binding toeach other.

The inventors have demonstrated that a truly modular DNA recognizing andpreferably binding polypeptide can be efficiently produced, wherein thebinding motif of said polypeptide is a repeat domain comprised of repeatunits which are selected on the basis of their recognition capability ofa combination of particular base pairs. Accordingly, it should be wellwithin the capability of one of normal skill in the art to design apolypeptide capable of binding to any desired target DNA sequence simplyby considering the sequence of base pairs present in the target DNA andcombining in the appropriate order repeat units as binding motifs havingthe necessary characteristics to bind thereto. The greater the length ofknown sequence of the target DNA, the greater the number of modularrepeat units that can be included in the polypeptide. For example, ifthe known sequence is only 9 bases long, then nine repeat units asdefined above can be included in the polypeptide. If the known sequenceis 27 bases long, then up to 27 repeat units could be included in thepolypeptide. The longer the target DNA sequence, the lower theprobability of its occurrence in any other given portion of DNAelsewhere in the genome.

Moreover, those repeat units selected for inclusion in the polypeptidecould be artificially modified in order to modify their bindingcharacteristics. Alternatively (or additionally) the length and aminoacid sequence of the repeat unit could be varied as long as its bindingcharacteristic is not affected.

Generally, it will be preferred to select those repeat units having highaffinity and high specificity for the target DNA sequence.

As described herein, effectors can be designed to recognize any suitabletarget site, for regulation of expression of any endogenous gene ofchoice. Examples of endogenous genes suitable for regulation includeVEGF, CCR5, ER.alpha., Her2/Neu, Tat, Rev, HBV C, S, X, and P, LDL-R,PEPCK, CYP7, Fibrinogen, ApoB, Apo E, Apo(a), renin, NF-.kappa.B,I-.kappa.B, TNF-.alpha., FAS ligand, amyloid precursor protein, atrialnaturetic factor, ob-leptin, ucp-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-12, G-CSF, GM-CSF, Epo, PDGF, PAF, p53, Rb, fetal hemoglobin,dystrophin, eutrophin, GDNF, NGF, IGF-1, VEGF receptors fit and flk,topoisomerase, telomerase, bcl-2, cyclins, angiostatin, IGF, ICAM-1,STATS, c-myc, c-myb, TH, PTI-1, polygalacturonase, EPSP synthase,FAD2-1, delta-12 desaturase, delta-9 desaturase, delta-15 desaturase,acetyl-CoA carboxylase, acyl-ACP-thioesterase, ADP-glucosepyrophosphorylase, starch synthase, cellulose synthase, sucrosesynthase, senescence-associated genes, heavy metal chelators, fatty acidhydroperoxide lyase, viral genes, protozoal genes, fungal genes, andbacterial genes. In general, suitable genes to be regulated includecytokines, lymphokines, growth factors, mitogenic factors, chemotacticfactors, onco-active factors, receptors, potassium channels, G-proteins,signal transduction molecules, disease resistance genes, and otherdisease-related genes.

In another aspect, a method of modulating expression of a target gene ina cell is provided. The cell may be preferably a plant cell, a humancell, animal cell, fungal cell or any other living cell. The cellscontain a polypeptide wherein said polypeptide comprises at least arepeat domain comprising repeat units, and these repeat units contain ahypervariable region and each repeat unit is responsible for therecognition of 1 base pair in said target DNA sequence. Said polypeptideis introduced either as DNA encoding for the polypeptide or thepolypeptide is introduced per se into the cell by methods known in theart. Regardless of how introduced, the polypeptide should include atleast one repeat domain that specifically recognizes and preferablybinds to a target DNA sequence of base pairs and modulates theexpression of a target gene. In a preferred embodiment, all repeat unitscontain a hypervariable region which determines recognition of basepairs in a target DNA sequence.

Examples of peptide sequences which can be linked to an effector of thepresent invention, for facilitating uptake of effectors into cells,include, but are not limited to: an 11 animo acid peptide of the tatprotein of HIV; a 20 residue peptide sequence which corresponds to aminoacids 84 103 of the p16 protein (see Fahraeus et al. (1996) CurrentBiology 6:84); the third helix of the 60-amino acid long homeodomain ofAntennapedia (Derossi et al. (1994) J. Biol. Chem. 269:10444); the hregion of a signal peptide such as the Kaposi fibroblast growth factor(K-FGF) h region; or the VP22 translocation domain from HSV (Elliot &O'Hare (1997) Cell 88:223 233). Other suitable chemical moieties thatprovide enhanced cellular uptake may also be chemically linked toeffectors.

Toxin molecules also have the ability to transport polypeptides acrosscell membranes. Often, such molecules are composed of at least two parts(called “binary toxins”): a translocation or binding domain orpolypeptide and a separate toxin domain or polypeptide. Typically, thetranslocation domain or polypeptide binds to a cellular receptor, andthen the toxin is transported into the cell. Several bacterial toxins,including Clostridium perfringens iota toxin, diphtheria toxin (DT),Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracistoxin, and pertussis adenylate cyclase (CYA), have been used in attemptsto deliver peptides to the cell cytosol as internal or amino-terminalfusions (Arora et al. (1993) J. Biol. Chem. 268:3334 3341; Perelle etal. (1993) Infect. Immun. 61:5147 5156 (1993); Stenmark et al. (1991) J.Cell Biol. 113:1025 1032 (1991); Donnelly et al. (1993) Proc. Natl.Acad. Sci. USA 90:3530 3534; Carbonetti et al. (1995) Abstr. Annu. Meet.Am. Soc. Microbiol. 95:295; Sebo et al. (1995) Infect. Immun. 63:38513857; Klimpel et al. (1992) Proc. Natl. Acad. Sci. USA 89:10277 10281;and Novak et al. (1992) J. Biol. Chem. 267:17186 17193).

Effectors can also be introduced into an animal cell, preferably amammalian cell, via liposomes and liposome derivatives such asimmunoliposomes. The term “liposome” refers to vesicles comprised of oneor more concentrically ordered lipid bilayers, which encapsulate anaqueous phase. The aqueous phase typically contains the compound to bedelivered to the cell, in this case an effector. The liposome fuses withthe plasma membrane, thereby releasing the effector into the cytosol.Alternatively, the liposome is phagocytosed or taken up by the cell in atransport vesicle. Once in the endosome or phagosome, the liposomeeither degrades or fuses with the membrane of the transport vesicle andreleases its contents.

The invention particularly relates to the field of plant andagricultural technology. In one aspect, the present invention isdirected to a method to modulate the expression of a target gene inplant cells, which method comprises providing plant cells with apolypeptide modified according to the invention, said polypeptide beingcapable of specifically recognizing a target nucleotide sequence, or acomplementary strand thereof, within a target gene, and allowing saidpolypeptide to recognize and particularly bind to said target nucleotidesequence, whereby the expression of said target gene in said plant cellsis modulated.

The polypeptide can be provided to the plant cells via any suitablemethods known in the art. For example, the protein can be exogenouslyadded to the plant cells and the plant cells are maintained underconditions such that the polypeptide is introduced into the plant cell,binds to the target nucleotide sequence and regulates the expression ofthe target gene in the plant cells. Alternatively, a nucleotidesequence, e.g., DNA or RNA, encoding the polypeptide can be expressed inthe plant cells and the plant cells are maintained under conditions suchthat the expressed polypeptide binds to the target nucleotide sequenceand regulates the expression of the target gene in the plant cells.

A preferred method to modulate the expression of a target gene in plantcells comprises the following steps: a) providing plant cells with anexpression system for a polypeptide modified according to the invention,said polypeptide being capable of specifically recognizing, andpreferably binding, to a target nucleotide sequence, or a complementarystrand thereof, within an expression control element of a target gene,preferably a promoter; and b) culturing said plant cells underconditions wherein said polypeptide is produced and binds to said targetnucleotide sequence, whereby expression of said target gene in saidplant cells is modulated.

Any target nucleotide sequence can be modulated by the present method.For example, the target nucleotide sequence can be endogenous orexogenous to the target gene. In an embodiment of the invention thetarget nucleotide sequence can be present in a living cell or present invitro. In a specific embodiment, the target nucleotide sequence isendogenous to the plant. The target nucleotide sequence can be locatedin any suitable place in relation to the target gene. For example, thetarget nucleotide sequence can be upstream or downstream of the codingregion of the target gene. Alternatively, the target nucleotide sequenceis within the coding region of the target gene. Preferably, the targetnucleotide sequence is a promoter of a gene.

Any target gene can be modulated by the present method. For example, thetarget gene can encode a product that affects biosynthesis,modification, cellular trafficking, metabolism and degradation of apeptide, a protein, an oligonucleotide, a nucleic acid, a vitamin, anoligosaccharide, a carbohydrate, a lipid, or a small molecule.Furthermore, effectors can be used to engineer plants for traits such asincreased disease resistance, modification of structural and storagepolysaccharides, flavors, proteins, and fatty acids, fruit ripening,yield, color, nutritional characteristics, improved storage capability,and the like.

Therefore, the invention provides a method of altering the expression ofa gene of interest in a target cell, comprising: determining (ifnecessary) at least part of the DNA sequence of the structural regionand/or a regulatory region of the gene of interest; designing apolypeptide including the repeat units modified in accordance with theinvention to recognize specific base pairs on the DNA of known sequence,and causing said modified polypeptide to be present in the target cell,(preferably in the nucleus thereof). (It will be apparent that the DNAsequence need not be determined if it is already known.)

The regulatory region could be quite remote from the structural regionof the gene of interest (e.g. a distant enhancer sequence or similar).

In addition, the polypeptide may advantageously comprise functionaldomains from other proteins (e.g. catalytic domains from restrictionendonucleases, recombinases, replicases, integrases and the like) oreven “synthetic” effector domains. The polypeptide may also compriseactivation or processing signals, such as nuclear localisation signals.These are of particular usefulness in targeting the polypeptide to thenucleus of the cell in order to enhance the binding of the polypeptideto an intranuclear target (such as genomic DNA).

The modified polypeptide may be synthesised in situ in the cell as aresult of delivery to the cell of DNA directing expression of thepolypeptide. Methods of facilitating delivery of DNA are well-known tothose skilled in the art and include, for example, recombinant viralvectors (e.g. retroviruses, adenoviruses), liposomes and the like.Alternatively, the modified polypeptide could be made outside the celland then delivered thereto. Delivery could be facilitated byincorporating the polypeptide into liposomes etc. or by attaching thepolypeptide to a targeting moiety (such as the binding portion of anantibody or hormone molecule, or a membrane transition domain, or thetranslocation domain of a fungal or oomycete effector, or thecell-binding B-domain of the classical A-B family of bacterial toxins).Indeed, one significant advantage of the modified proteins of theinvention in controlling gene expression would be the vector-freedelivery of protein to target cells.

To the best knowledge of the inventors, design of a polypeptidecontaining modified repeat units capable of specifically recognizingbase pairs in a target DNA sequence and its successful use in modulationof gene expression (as described herein) has never previously beendemonstrated. Thus, the breakthrough of the present invention asdisclosed herein presents numerous possibilities that extend beyond usesin plants. In one embodiment of the invention, effector polypeptides aredesigned for therapeutic and/or prophylactic use in regulating theexpression of disease-associated genes. For example, said polypeptidescould be used to inhibit the expression of foreign genes (e.g., thegenes of bacterial or viral pathogens) in humans, other animals, orplants, or to modify the expression of mutated host genes (such asoncogenes).

The invention therefore also provides an effector polypeptide capable ofinhibiting the expression of a disease-associated gene. Typically thepolypeptide will not be a naturally occurring polypeptide but will bespecifically designed to inhibit the expression of thedisease-associated gene. Conveniently the effector polypeptide will bedesigned by any of the methods of the invention.

The invention also relates to the field of genome engineering. Aneffector polypeptide can be generated according to the invention totarget a specific DNA sequence in a genome. Said polypeptide can bemodified to contain an activity that directs modification of the targetDNA sequence (e.g. site specific recombination or integration of targetsequences). This method enables targeted DNA modifications in complexgenomes.

In a still further embodiment of the invention, a polypeptide isprovided which is modified to include at least a repeat domaincomprising repeat units, the repeat units having hypervariable regionfor determining selective recognition of a base pair in a DNA sequence.

In a preferred embodiment, the polypeptide comprises within said repeatunit a hypervariable region which is selected from the following groupin order to determine recognition of one of the following base pairs:

-   -   HD for recognition of C/G    -   NI for recognition of A/T    -   NG for recognition of T/A    -   NS for recognition of C/G or A/T or T/A or G/C    -   NN for recognition of G/C or A/T    -   IG for recognition of T/A    -   N for recognition of C/G or T/A    -   HG for recognition of T/A    -   H for recognition of T/A    -   NK for recognition of G/C    -   NH for recognition of G/C    -   NP for recognition of A/T or C/G or T/A    -   NT for recognition of A/T or G/C    -   HN for recognition of A/T or G/C    -   SH for recognition of G/C    -   SN for recognition of G/C and    -   IS for recognition of A/T.

The polypeptides of the present invention can further comprise within arepeat unit a hypervariable region which is selected from the followinggroup in order to determine recognition of one of the following basepairs: HA for recognition of C/G; ND for recognition of C/G; HI forrecognition of C/G; FIN for recognition of G/C; and NA for recognitionof G/C.

The invention also comprises DNA which encodes for any one of thepolypeptides described before.

In a still further embodiment, DNA is provided which is modified toinclude a base pair located in a target DNA sequence so that said basepair can be specifically recognized by a polypeptide which includes atleast a repeat domain comprising repeat units, the repeat units having ahypervariable region which determine recognition of said base pair insaid DNA. In one optional embodiment, said base pair is located in agene expression control sequence. Due to the modular assembly of therepeat domain, a sequence of base pairs can be specifically targeted bysaid repeat domain.

In an alternative embodiment of the invention, said DNA is modified by abase pair selected from the following group in order to receive aselective and determined recognition by one of the followinghypervariable regions:

-   -   C/G for recognition by HD    -   A/T for recognition by NI    -   T/A for recognition by NG    -   CT or A/T or T/A or G/C for recognition by NS    -   G/C or A/T for recognition by NN    -   T/A for recognition by IG.    -   C/G or T/A for recognition by N    -   T/A for recognition by HG    -   T/A for recognition by H    -   G/C for recognition by NK    -   G/C for recognition of NH    -   A/T or C/G or T/A for recognition of NP    -   A/T or G/C for recognition of NT    -   A/T or G/C for recognition of HN    -   G/C for recognition of SH    -   G/C for recognition of SN and    -   A/T for recognition of IS.

The DNA of the present invention can be modified to modified by a basepair selected from the following group in order to receive a selectiveand determined recognition by one of the following hypervariableregions: HA for recognition of C/G; ND for recognition of C/G; HI forrecognition of C/G; HN for recognition of G/C; and NA for recognition ofG/C.

In yet another aspect the invention provides a method of modifying anucleic acid sequence of interest present in a sample mixture by bindingthereto a polypeptide according to the invention, comprising contactingthe sample mixture with said polypeptide having affinity for at least aportion of the sequence of interest, so as to allow the polypeptide torecognize and preferably bind specifically to the sequence of interest.

The term “modifying” as used herein is intended to mean that thesequence is considered modified simply by the binding of thepolypeptide. It is not intended to suggest that the sequence ofnucleotides is changed, although such changes (and others) could ensuefollowing binding of the polypeptide to the nucleic acid of interest.Conveniently the nucleic acid sequence is DNA.

Modification of the nucleic acid of interest (in the sense of bindingthereto by a polypeptide modified to contain modular repeat units) couldbe detected in any of a number of methods (e.g. gel mobility shiftassays, use of labelled polypeptides—labels could include radioactive,fluorescent, enzyme or biotin/streptavidin labels).

Modification of the nucleic acid sequence of interest (and detectionthereof) may be all that is required (e.g. in diagnosis of disease).Desirably, however, further processing of the sample is performed.Conveniently the polypeptide (and nucleic acid sequences specificallybound thereto) is separated from the rest of the sample. Advantageouslythe polypeptide-DNA complex is bound to a solid phase support, tofacilitate such separation. For example, the polypeptide may be presentin an acrylamide or agarose gel matrix or, more preferably, isimmobilised on the surface of a membrane or in the wells of a microtitreplate.

In one embodiment of the invention, said repeat domain comprising repeatunits is inserted in a bacterial, viral, fungal, oomycete, human, animalor plant polypeptide to achieve a targeted recognition and preferablybinding of one or more specified base pairs in a DNA sequence, andoptionally wherein said repeat units are taken from the repeat domainsof AvrBs3-like family of proteins which are further optionally modifiedin order to obtain a pre-selected specific binding activity to one ormore base pairs in a DNA sequence.

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments and variants of the disclosed DNA sequences and proteinsencoded thereby are also encompassed by the present invention. By“fragment” is intended a portion of the DNA sequence or a portion of theamino acid sequence and hence protein encoded thereby. Fragments of aDNA sequence comprising coding sequences may encode protein fragmentsthat retain biological activity of the native protein and hence DNArecognition or binding activity to a target DNA sequence as hereindescribed. Alternatively, fragments of a DNA sequence that are useful ashybridization probes generally do not encode proteins that retainbiological activity or do not retain promoter activity. Thus, fragmentsof a DNA sequence may range from at least about 20 nucleotides, about 50nucleotides, about 100 nucleotides, and up to the full-lengthpolynucleotide of the invention.

“Variants” is intended to mean substantially similar sequences. For DNAsequences, a variant comprises a DNA sequence having deletions (i.e.,truncations) at the 5′ and/or 3′ end; deletion and/or addition of one ormore nucleotides at one or more internal sites in the nativepolynucleotide; and/or substitution of one or more nucleotides at one ormore sites in the native polynucleotide. As used herein, a “native” DNAsequence or polypeptide comprises a naturally occurring DNA sequence oramino acid sequence, respectively. For DNA sequences, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the polypeptidesof the invention. Variant DNA sequences also include syntheticallyderived DNA sequences, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a protein of theinvention. Generally, variants of a particular DNA sequence of theinvention will have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters as described elsewhere herein.

Variants of a particular DNA sequence of the invention (i.e., thereference DNA sequence) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantDNA sequence and the polypeptide encoded by the reference DNA sequence.Percent sequence identity between any two polypeptides can be calculatedusing sequence alignment programs and parameters described elsewhereherein. Where any given pair of polynucleotides of the invention isevaluated by comparison of the percent sequence identity shared by thetwo polypeptides they encode, the percent sequence identity between thetwo encoded polypeptides is at least about 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion (so-called truncation) of one or more amino acids atthe N-terminal and/or C-terminal end of the native protein; deletionand/or addition of one or more amino acids at one or more internal sitesin the native protein; or substitution of one or more amino acids at oneor more sites in the native protein. Variant proteins encompassed by thepresent invention are biologically active, that is they continue topossess the desired biological activity of the native protein asdescribed herein. Such variants may result from, for example, geneticpolymorphism or from human manipulation. Biologically active variants ofa protein of the invention will have at least about 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the amino acid sequence for the native protein as determinedby sequence alignment programs and parameters described elsewhereherein. A biologically active variant of a protein of the invention maydiffer from that protein by as few as 1-15 amino acid residues, as fewas 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 aminoacid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants and fragments of the proteins canbe prepared by mutations in the DNA. Methods for mutagenesis andpolynucleotide alterations are well known in the art. See, for example,Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al.(1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walkerand Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.), herein incorporated by reference.Conservative substitutions, such as exchanging one amino acid withanother having similar properties, may be optimal.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays as described elsewhere hereinor known in the art.

Variant DNA sequences and proteins also encompass sequences and proteinsderived from a mutagenic and recombinogenic procedure such as DNAshuffling. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

In a PCR approaches, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the DNA sequences of the invention.Methods for preparation of probes for hybridization and for constructionof cDNA and genomic libraries are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)-500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

It is recognized that the DNA sequences and proteins of the inventionencompass polynucleotide molecules and proteins comprising a nucleotideor an amino acid sequence that is sufficiently identical to the DNAsequences or to the amino acid sequence disclosed herein. The term“sufficiently identical” is used herein to refer to a first amino acidor nucleotide sequence that contains a sufficient or minimum number ofidentical or equivalent (e.g., with a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences have acommon structural domain and/or common functional activity. For example,amino acid or nucleotide sequences that contain a common structuraldomain having at least about 70% identity, preferably 75% identity, morepreferably 85%, 90%, 95%, 96%, 97%, 98% or 99% identity are definedherein as sufficiently identical.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, nonlimitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to the polynucleotide molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0), which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the full-length sequences ofthe invention and using multiple alignment by mean of the algorithmClustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using theprogram AlignX included in the software package Vector NTI Suite Version7 (InforMax, Inc., Bethesda, Md., USA) using the default parameters; orany equivalent program thereof. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by CLUSTALW (Version 1.83) usingdefault parameters (available at the European Bioinformatics Institutewebsite: http://www.ebi.ac.uk/Tools/clustalw/html).

The DNA sequences of the invention can be provided in expressioncassettes for expression in any prokaryotic or eukaryotic cell and/ororganism of interest including, but not limited to, bacteria, fungi,algae, plants, and animals. The cassette will include 5′ and 3′regulatory sequences operably linked to a DNA sequence of the invention.“Operably linked” is intended to mean a functional linkage between twoor more elements. For example, an operable linkage between apolynucleotide or gene of interest and a regulatory sequence (i.e., apromoter) is functional link that allows for expression of thepolynucleotide of interest. Operably linked elements may be contiguousor non-contiguous. When used to refer to the joining of two proteincoding regions, by operably linked is intended that the coding regionsare in the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes. Such an expression cassette is provided with aplurality of restriction sites and/or recombination sites for insertionof the DNA sequence to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in plants or other organism or non-human host cell.The regulatory regions (i.e., promoters, transcriptional regulatoryregions, and translational termination regions) and/or the DNA sequenceof the invention may be native/analogous to the host cell or to eachother. Alternatively, the regulatory regions and/or DNA sequence of theinvention may be heterologous to the host cell or to each other. As usedherein, “heterologous” in reference to a sequence is a sequence thatoriginates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide. Asused herein, a chimeric gene comprises a coding sequence operably linkedto a transcription initiation region that is heterologous to the codingsequence.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the host, or may be derived from another source(i.e., foreign or heterologous) to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof. Convenienttermination regions for use in plants are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increasedexpression in a transformed organism. That is, the polynucleotides canbe synthesized using codons preferred by the host for improvedexpression. See, for example, Campbell and Gowri (1990) Plant Physiol.92:1-11 for a discussion of host-preferred codon usage. Methods areavailable in the art for synthesizing host-preferred gene, particularlyplant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498,herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa etal. (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the host of interest and the desiredoutcome. The nucleic acids can be combined with constitutive,tissue-preferred, or other promoters for expression in plants. Suchconstitutive promoters include, for example, the core CaMV 35S promoter(Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al.(1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) PlantMol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689); pEMU (Last et al. (1991) Theon. Appl. Genet. 81:581-588);MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat.No. 5,659,026), and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced expressionwithin a particular host tissue. Such tissue-preferred promoters for usein plants include, but are not limited to, leaf-preferred promoters,root-preferred promoters, seed-preferred promoters, and stem-preferredpromoters. Tissue-preferred promoters include Yamamoto et al. (1997)Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343;Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) PlantPhysiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozcoet al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993)Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al.(1993) Plant J. 4(3):495-505. Such promoters can be modified, ifnecessary, for weak expression.

Generally, it will be beneficial to express the gene from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819, herein incorporated byreference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant. Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

Numerous plant transformation vectors and methods for transformingplants are available. See, for example, An, G. et al. (1986) PlantPysiol., 81:301-305; Fry, J., et al. (1987) Plant Cell Rep. 6:321-325;Block, M. (1988) Theon. Appl Genet. 76:767-774; Hinchee, et al. (1990)Stadler. Genet. Symp. 203212.203-212; Cousins, et al. (1991) Aust. J.Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene.118:255-260; Christou, et al. (1992) Trends. Biotechnol. 10:239-246;D'Halluin, et al. (1992) Bio/Technol. 10:309-314; Dhir, et al. (1992)Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad. Sci. USA90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant;29P:119-124; Davies, et al. (1993) Plant Cell Rep. 12:180-183; Dong, J.A. and Mchughen, A. (1993) Plant Sci. 91:139-148; Franklin, C. I. andTrieu, T. N. (1993) Plant. Physiol. 102:167; Golovkin, et al. (1993)Plant Sci. 90:41-52; Guo Chin Sci. Bull. 38:2072-2078; Asano, et al.(1994) Plant Cell Rep. 13; Ayeres N. M. and Park, W. D. (1994) Crit.Rev. Plant. Sci. 13:219-239; Barcelo, et al. (1994) Plant. J. 5:583-592;Becker, et al. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta.Physiol Plant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech.5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman, etal. (1994) Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol.Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol.104:3748.

The methods of the invention involve introducing a polynucleotideconstruct comprising a DNA sequence into a host cell. By “introducing”is intended presenting to the plant the polynucleotide construct in sucha manner that the construct gains access to the interior of the hostcell. The methods of the invention do not depend on a particular methodfor introducing a polynucleotide construct into a host cell, only thatthe polynucleotide construct gains access to the interior of one cell ofthe host. Methods for introducing polynucleotide constructs intobacteria, plants, fungi and animals are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “stable transformation” is intended that the polynucleotide constructintroduced into a plant integrates into the genome of the host and iscapable of being inherited by progeny thereof. By “transienttransformation” is intended that a polynucleotide construct introducedinto the host does not integrate into the genome of the host.

For the transformation of plants and plant cells, the DNA sequences ofthe invention are inserted using standard techniques into any vectorknown in the art that is suitable for expression of the DNA sequences ina host cell or organism of interest. The selection of the vector dependson the preferred transformation technique and the target host species tobe transformed.

Methodologies for constructing plant expression cassettes andintroducing foreign nucleic acids into plants are generally known in theart and have been previously described. For example, foreign DNA can beintroduced into plants, using tumor-inducing (Ti) plasmid vectors. Othermethods utilized for foreign DNA delivery involve the use of PEGmediated protoplast transformation, electroporation, microinjectionwhiskers, and biolistics or microprojectile bombardment for direct DNAuptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 toVasil et al.; Bilang et al. (1991) Gene 100: 247-250; Scheid et al.,(1991) Mol. Gen. Genet., 228: 104-112; Guerche et al., (1987) PlantScience 52: 111-116; Neuhause et al., (1987) Theor. Appl Genet. 75:30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980)Science 208:1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlocket al., (1989) Plant Physiology 91: 694-701; Methods for Plant MolecularBiology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) andMethods in Plant Molecular Biology (Schuler and Zielinski, eds.)Academic Press, Inc. (1989). The method of transformation depends uponthe plant cell to be transformed, stability of vectors used, expressionlevel of gene products and other parameters.

The DNA sequences of the invention may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a polynucleotide construct of theinvention within a viral DNA or RNA molecule. It is recognized that thea protein of the invention may be initially synthesized as part of aviral polyprotein, which later may be processed by proteolysis in vivoor in vitro to produce the desired recombinant protein. Further, it isrecognized that promoters of the invention also encompass promotersutilized for transcription by viral RNA polymerases. Methods forintroducing polynucleotide constructs into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

In specific embodiments, the DNA sequences of the invention can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the protein or variants and fragments thereofdirectly into the plant or the introduction of a transcript encoding theprotein into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol. Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci.44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107:775-784, all of whichare herein incorporated by reference. Alternatively, the polynucleotidecan be transiently transformed into the plant using techniques known inthe art. Such techniques include Agrobacterium tumefaciens-mediatedtransient expression as described below.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide construct ofthe invention, for example, an expression cassette of the invention,stably incorporated into their genome.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Plants ofparticular interest include, but are not limited to, and grain plantsthat provide seeds of interest, oil-seed plants, leguminous plants, andArabidopsis thaliana. Seeds of interest include grain seeds, such ascorn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants includecotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm,coconut, etc. Leguminous plants include beans and peas. Beans includeguar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean,lima bean, fava bean, lentils, chickpea, etc.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruits, roots, root tips, anthers, and the like. Progeny,variants, and mutants of the regenerated plants are also included withinthe scope of the invention, provided that these parts comprise theintroduced polynucleotides.

The present invention further encompasses the introduction of the DNAsequences of the invention into non-plant host cells, including, but notlimited to, bacterial cells, yeast cells other fungal cells, humancells, and other animal cells. In addition, the invention encompassesthe introduction of the DNA sequences into animals and other organismsby both stable and transient transformation methods.

As discussed herein, a DNA sequence of the present invention can beexpressed in these eukaryotic systems. Synthesis of heterologouspolynucleotides in yeast is well known (Sherman et al. (1982) Methods inYeast Genetics, Cold Spring Harbor Laboratory). Two widely utilizedyeasts for production of eukaryotic proteins are Saccharomycescerevisiae and Pichia pastoris. Vectors, strains, and protocols forexpression in Saccharomyces and Pichia are known in the art andavailable from commercial suppliers (e.g., Invitrogen). Suitable vectorsusually have expression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of mammalian orinsect origin. Illustrative cell cultures useful for the production ofthe peptides are mammalian cells. A number of suitable host cell linescapable of expressing intact proteins have been developed in the art,and include the HEK293, BHK21, and CHO cell lines. Expression vectorsfor these cells can include expression control sequences, such as anorigin of replication, a promoter (e.g. the CMV promoter, a HSV tkpromoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queenet al. (1986) Immunol. Rev. 89:49), and necessary processing informationsites, such as ribosome binding sites, RNA splice sites, polyadenylationsites (e.g., an SV40 large T Ag poly A addition site), andtranscriptional terminator sequences. Other animal cells useful forproduction of proteins of the present invention are available, forinstance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See, Schneider(1987) J. Embyol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP 1 intron from SV40 (Sprague et al.(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors (Saveria-Campo (1985)DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press,Arlington, Va., pp. 213-238).

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextrin, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art (Kuchler(1997) Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc.).

Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used in the method ofthe invention. Commonly used prokaryotic control sequences which aredefined herein to include promoters for transcription initiation,optionally with an operator, along with ribosome binding sequences,include such commonly used promoters as the beta lactamase(penicillinase) and lactose (lac) promoter systems (Chang et al. (1977)Nature 198:1056), the tryptophan (trp) promoter system (Goeddel et al.(1980) Nucleic Acids Res. 8:4057) and the lambda derived P L promoterand N-gene ribosome binding site (Shimatake et al. (1981) Nature292:128). The inclusion of selection markers in DNA vectors transfectedin E coli. is also useful. Examples of such markers include genesspecifying resistance to ampicillin, tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235); Mosbach et al. (1983) Nature 302:543-545).

With respect to fusion proteins, “operably linked” is intended to mean afunctional linkage between two or more elements or domains. If itrecognized that a linker of one or more amino acids may be inserted inbetween each of the two or more elements to maintain the desiredfunction of the two or more elements.

In one embodiment of the invention, fusion proteins comprise a repeatdomain of the invention operably linked to at least one protein or partor domain thereof. In certain embodiments of the invention, the proteinor part or domain thereof comprises a protein or functional part ordomain thereof, that is capable of modifying DNA or RNA. In otherembodiments, protein or functional part or domain thereof is capable offunctioning as a transcriptional activator or a transcriptionalrepressor. Preferred proteins include, but are not limited to,transcription activators, a transcription repressors, aresistance-mediating proteins, nucleases, topoisomerases, ligases,integrases, recombinases, resolvases, methylases, acetylases,demethylases, and deacetylases.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Identification of the Basis for DNA Specificity ofTAL Effectors

The fact that AvrBs3 directly binds the UPA-box, a promoter element ininduced target genes (Kay et al. (2007) Science 318, 648-651; Römer etal. (2007) Science 318:645-648), prompted us to investigate the basisfor DNA-sequence specificity. Each repeat region generally consists of34 amino acid, and the repeat units are nearly identical; however, aminoacids 12 and 13 are hypervariable (Schornack et al. (2006) J. PlantPhysiol. 163:256-272; FIG. 1A). The most C-terminal repeat of AvrBs3shows sequence similarity to other repeat units only in its first 20amino acids and is therefore referred to as half repeat. The repeatunits can be classified into different repeat types based on theirhypervariable 12th and 13th amino acids (FIG. 1B). Because the size ofthe UPA-box (18 (20)/19 (21) bp) almost corresponds to the number ofrepeat units (17.5) in AvrBs3, we considered the possibility that onerepeat unit of AvrBs3 contacts one specific DNA base pair. When therepeat types of AvrBs3 (amino acid 12 and 13 of each repeat) areprojected onto the UPA box, it becomes evident that certain repeat typescorrelate with specific base pairs in the target DNA. For example, HDand NI repeat units have a strong preference for C and A, respectively(FIG. 1B). For simplicity, we designate only bases in the upper (sense)DNA strand. Our model of recognition specificity is supported by thefact that the AvrBs3 repeat deletion derivative AvrBs3Δrep16 which lacksfour repeat units (Δ11-14; FIG. 5A, B) recognizes a shorter anddifferent target DNA sequence (FIGS. 5 to 8). Based on sequencecomparisons of UPA-boxes of AvrBs3-induced pepper genes and mutationalanalysis, the target DNA box of AvrBs3 appears to be 1 bp longer thanthe number of repeat units in AvrBs3. In addition, a T is conserved atthe 5′ end of the UPA box immediately preceding the predictedrecognition specificity of the first repeat (FIG. 1). Intriguingly,secondary structure predictions of the protein region preceding thefirst repeat and the repeat region show similarities, despite lack ofamino acid-sequence conservation. This suggests an additional repeat,termed repeat 0 (FIG. 1B).

To further substantiate and extend our model (FIG. 1B), we predicted theyet unknown target DNA sequences of Xanthomonas TAL effectors based onthe sequence of their repeat units, and inspected the promoters of knownTAL target genes and their alleles for the presence of putative bindingsites. We identified sequences matching the predicted specificity inpromoters of alleles that are induced in response to the correspondingTAL effector, but not in non-induced alleles

(FIGS. 5C-F). The presence of these boxes suggests that the inducedgenes are direct targets of the corresponding TAL effectors. Based onthe DNA base frequency for different repeat types in the target DNAsequences using eight TAL effectors we deduced a code for the DNA targetspecificity of certain repeat types (FIG. 1C, D; FIG. 5).

To experimentally validate our model we predicted target DNA sequencesfor the TAL effectors Hax2 (21.5 repeat units), Hax3 (11.5 repeatunits), and Hax4 (14.5 repeat units) from the Brassicaceae-pathogen X.campestris pv. armoraciae (22). First, we derived target DNA boxes forHax3 and Hax4, because they exclusively contain repeat-types present inAvrBs3 (amino acid 12/13: NI, HD, NG, NS; FIG. 1A, FIG. 2A) for whichDNA binding and gene activation have been shown experimentally. The Hax3and Hax4 target boxes were placed in front of the minimal (−55 to +25)tomato Bs4 promoter, which has very weak basal activity (Schornack etal. (2005) Mol. Plant-Microbe Interact. 18:1215-1225; FIG. 2B; FIG. 9),driving a promoterless uidA (β-glucuronidase, GUS) reporter gene. Fortransient expression studies, we transfected the reporter constructstogether with cauliflower mosaic virus 35S-promoter driven effectorgenes hax3 and hax4 into Nicotiana benthamiana leaves usingAgrobacterium-mediated T-DNA delivery. Qualitative and quantitative GUSassays demonstrated that promoters containing the Hax3- or Hax4-box werestrongly and specifically induced in the presence of the correspondingeffector (FIG. 2C). Likewise, we addressed the importance of the firstnucleotide (T) in the predicted target DNA sequence of Hax3 andgenerated four different Hax3-boxes with either A, C, G or T at the 5′end (FIG. 10A, B). Coexpression of hax3 and the reporter constructs inN. benthamiana demonstrated that only a promoter containing a Hax3-boxwith a 5′ T was strongly induced in the presence of Hax3 whereas theothers led to weaker activation (FIG. 10C). This indicates that position0 contributes to promoter activation specificity of Hax3 and likelyother TAL effectors. To address the possibility that some repeat typesconfer broader specificity, i.e., recognize more than one base, wepermutated the Hax4-box (FIG. 3A, B). Transient GUS assays showed thatNI-, HD-, and NG-repeat units in Hax4 strongly favour recognition of thebases A, C, and T, respectively, whereas NS-repeat units recognize allfour bases (FIG. 3B; FIG. 11). As several TAL effectors containNN-repeat units (FIG. 5 and FIG. 15, Table 1), we generated ArtX1, anartificial TAL effector with NN-repeat units and deduced a correspondingDNA recognition sequence using our code (FIG. 3C). Analysis of ArtX1-boxderivatives demonstrated that NN-repeat units recognize both A and G,with preference for G (FIG. 3C). This result confirms our prediction ofthe natural AvrXa27-box in rice which contains either an A or a G atpositions corresponding to NN-repeat units (FIG. 5C). In addition, wederived two possible AvrXa10-boxes with either A or G at positionscorresponding to NN-repeat units in AvrXa10. Both reporter constructswere induced efficiently by AvrXa10 (FIG. 12). Together, these datastrongly suggest that some repeat types recognize specific base pairswhereas others are more flexible.

An exceptional TAL effector is Hax2 because it contains 35 amino acidsper repeat instead of the typical 34 amino acid-repeat units (Kay et al.(2005) Mol. Plant-Microbe Interact. 18:838-848). In addition, Hax2contains a rare amino acid combination in its second repeat (amino acids12/13: IG; FIG. 2A). We permutated the corresponding third base of theHax2-box and analyzed reporter gene activation with the effector Hax2using the transient assay. This showed that an IG repeat confersspecificity for T (FIG. 13). The Hax2-box only leads to promoteractivation by Hax2, but not by Hax3 or Hax4 (FIG. 2C). This demonstratesthat 35 amino acid-repeat units function like 34 amino acid-repeatunits. This is supported by the fact that the TAL effector AvrHah1 whichcontains 35 amino acid repeat units, induces Bs3-mediated resistance(Schornack et al. (2008) New Phytol. 179:546-556). The repeat types ofAvrHah1 match to the UPA-box in the Bs3 promoter (FIG. 5A, B).

Interestingly, the expression of hax2 in Arabidopsis thaliana leads topurple coloured leaves, indicating an accumulation of anthocyanin (FIGS.14A, B). To identify Hax2 target genes we analyzed promoter regions ofthe A. thaliana genome using pattern search (Patmatch, TAIR;www.arabidopsis.org) with degenerated Hax2-box sequences. One of theputative Hax2 target genes encodes the MYB transcription factor PAP1(At1G56650) which controls anthocyanin biosynthesis (Borevitz et al.(2000) Plant Cell 12:2383-2394). Semiquantitative analysis of the PAP1transcript level demonstrated that expression of PAP1 is stronglyinduced by Hax2 (FIG. 14C). Visual inspection of the PAP1 promoterregion revealed the presence of a suboptimal Hax2-box (FIGS. 14D, E).Based on the code for TAL effector repeat types (FIG. 1D) and the datadescribed above we predicted putative target DNA sequences foradditional TAL effectors some of which are important virulence factors(FIG. 15, Table 1).

Because the repeat number in TAL effectors ranges from 1.5 to 28.5, akey question is whether effectors with few repeat units can activategene expression. Therefore, we tested how the number of repeat unitsinfluences target gene expression. For this, we constructed artificialeffectors containing the N- and C-terminal regions of Hax3 and a repeatdomain with 0.5 to 15.5 HD-repeat units (specificity for C). Fortechnical reasons, the first repeat in all cases was NI (specificity forA). The corresponding target DNA box consists of 17 C-residues precededby TA (FIGS. 4A, B). Promoter activation by the artificial effectors wasmeasured using the transient Bs4-promoter GUS-assay in N. benthamiana.While at least 6.5 repeat units were needed for gene induction, 10.5 ormore repeat units led to strong reporter gene activation (FIG. 4C).These data demonstrate that a minimal number of repeat units is requiredto recognize the artificial target DNA-box and activate gene expression.The results also suggest that effectors with fewer repeat numbers arelargely inactive. We have shown that the repeat region of TAL effectorshas a sequential nature that corresponds to a consecutive target DNAsequence. Hence, it should be feasible to generate effectors with novelDNA-binding specificities. Three artificial effectors were generated(ArtX1, ArtX2, ArtX3), each with randomly assembled 12.5 repeat units(FIGS. 3C, D), and tested for induction of Bs4 promoter-reporter fusionscontaining predicted target DNA-sequences. All three artificialeffectors strongly and specifically induced the GUS reporter only inpresence of the corresponding target DNA-box (FIG. 3E; FIG. 11). Ourmodel for recognition specificity of TAL effectors in which one repeatunit contacts one base pair in the DNA via amino acids 12 and 13 of eachrepeat enables to predict the binding specificity of TAL effectors andidentification of plant target genes. As many TAL effectors are majorvirulence factors the knowledge of plant target genes will greatlyenhance our understanding of plant disease development caused byxanthomonads. In addition, we successfully designed artificial effectorsthat act as transcription factors with specific DNA-binding domains.Previously, zinc finger transcription factors containing a tandemarrangement of zinc finger units have been engineered to bind specifictarget DNA sequences.

Similarly, TAL effectors have a linear DNA-binding specificity that caneasily be rearranged. It has not escaped our notice that the postulatedright-handed superhelical structure of the repeat regions in TALeffectors immediately suggests a possible mechanism for interaction withthe right-handed helix of the genetic material. It will be important todetermine the structure of the novel DNA-binding domain of TAL effectorscomplexed with target DNA.

The following paragraphs describe further embodiments of the invention:

1) Prediction of DNA-Binding Specificities of Naturally OccurringAvrbs3-Homologous Proteins and Generation of Resistant Plants.

The repeat units of the repeat domain of naturally occurring effectorsof the AvrBs3-family encode a corresponding DNA-binding specificity.These recognition sequences can be predicted with the recognition code.

The artificial insertion of the predicted recognition sequences in frontof a gene in transgenic plants leads to expression of the gene if thecorresponding AvrBs3-like effector is translocated into the plant cell(e.g. during a bacterial infection).

If the recognition sequence is inserted in front of a gene whoseexpression leads to a defence reaction (resistance-mediating gene) ofthe plant, such constructed transgenic plants are resistant against aninfection of plant pathogenic bacteria which translocate thecorresponding effector.

2) The Identification of Plant Genes Whose Expression is Induced by aSpecific Effector of the AvrBs3-Family

The prediction of DNA target sequences of a corresponding effector ofthe AvrBs3-family in the promoter region of plant genes is an indicationfor the inducible expression of these genes by the effector. Using themethod according to the invention it is possible to predict inducibleplant genes. Predictions are particularly straightforward in sequencedgenomes.

3) Use of Other Effectors as Transcriptional Activators in ExpressionSystems

Analogous to the use of Hax3 and Hax4, the predicted DNA bindingsequences of other members of the AvrBs3-family can be inserted intopromoters to generate new controllable promoters which can be induced bythe corresponding effector.

4) Construction of a Secondarily Inducible System

Two constructs are introduced into plants. First, a hax3 gene whoseexpression is under control of an inducible promoter. Secondly, a targetgene that contains the Hax3-box in the promoter.

Induction of the expression of hax3 leads to production of the Hax3protein that then induces the expression of the target gene. Thedescribed two-component construction leads to a twofold expressionswitch which allows a variable expression of the target gene. Thetrans-activator and the target gene can also be present first indifferent plant lines and can be introgressed at will. Analogous tothis, Hax4 and the corresponding Hax4-box can be used. This system canalso be used with other members of the AvrBs3-family or artificialderivatives and predicted DNA-target sequences. The functionality of thesystem could already be verified. Transgenic Arabidopsis thaliana plantswere constructed, which contain an inducible avrBs3 gene as well as aBs3 gene under control of its native promoter, whose expression can beinduced by AvrBs3. The induction of expression of avrBs3 leads toexpression of Bs3 and therefore to cell death. See, WO 2009/042753,herein incorporated by reference.

5) Construction of Disease-Resistant Plants

If the DNA target sequence of an AvrBs3-similar effector is inserted infront of a gene whose expression leads to a defence reaction(resistance-mediating gene) of the plant, correspondingly constructedtransgenic plants will be resistant against infection of plantpathogenic organisms, which make this effector available. Such aresistance-mediating gene can for example lead to a local cell deathwhich prevents spreading of the organisms/pathogens, or induce the basalor systemic resistance of the plant cell.

6) Generation of Repeat Domains for the Detection of a Specific DNASequence and Induction of Transcription of Following Genes

The modular architecture of the central repeat domain enables thetargeted construction of definite DNA binding specificities and withthis the induction of transcription of selected plant genes. The DNAbinding specificities can either be artificially inserted in front oftarget genes so that novel effector-DNA-box variants are generated forthe inducible expression of target genes. Moreover, repeat domains canbe constructed that recognize a naturally occurring DNA sequence inorganisms. The advantage of this approach is that the expression of anygene in non-transgenic organisms can be induced if a correspondingeffector of the invention is present in the cells of this organism.

Introduction of the effector can be done in different ways:

(1) transfer via bacteria with a protein transport system (e.g. type-IIIsecretion system);(2) cell-bombardment with an artificial AvrBs3-protein;(3) transfer of a DNA-segment that leads to production of the effector,via introgression, Agrobacterium, viral vectors or cell-bombardment; or(4) other methods that result in uptake of the effector protein by thetarget cell

The central repeat domain of effectors of the AvrBs3-family is a newtype of DNA binding domain (Kay et al., 2007). The decryption of thespecificity of the single repeat units now allows the targetedadaptation of the DNA-binding specificity of this region. The DNAbinding region can be translationally fused to other functional domainsto generate sequence-specific effects. Below, four examples of suchprotein fusions are given.

7) Construction of Transcriptional Activators for the InducibleExpression of Genes in Cells of Living Organisms

The effectors of the AvrBs3-like family induce the expression of genesin plant cells. For this, the C-terminus of the protein is essential,which contains a transcriptional activation domain and nuclearlocalization sequences that mediate the import of the protein into theplant nucleus. The C-terminus of the AvrBs3-homologous protein can bemodified in such a way that it mediates the expression of genes infungal, animal, or human systems. Thereby, effectors can constructedthat function as transcriptional activators in humans, other animals, orfungi. Thus, the methods according to the invention can be applied notonly to plants, but also to other living organisms.

8) Use of Effectors as Transcriptional Repressors

The DNA binding specificity of the repeat domain can be used togetherwith other domains in protein fusions to construct effectors that act asspecific repressors. These effectors exhibit a DNA binding specificitythat has been generated in such a way that they bind to promoters oftarget genes. In contrast to the TAL effectors which are transcriptionactivators, these effectors are constructed to block the expression oftarget genes. Like classical repressors, these effectors are expected tocover promoter sequences by their recognition of, or binding to, atarget DNA sequence and make them inaccessible for factors thatotherwise control the expression of the target genes. Alternatively, orin addition, the repeat domains can be fused to atranscription-repressing domain, such as an EAR motif (Ohta et al. PlantCell 13:1959-1968 (2001)).

9) Use of Repeat Domains for Labelling and Isolation of SpecificSequences

The capability of a repeat domain to recognize a specific target DNAsequence an be used together with other domains to label specific DNAsequences. C-terminally a GFP (“green-fluorescent-protein”) can forexample be fused to an artificial repeat domain that detects a desiredDNA sequence. This fusion protein binds in vivo and in vitro to acorresponding DNA sequence. The position of this sequence on thechromosome can be localized using the fused GFP-protein. In an analogousway, other protein domains that enable a cellular localization of theprotein (e.g. by FISH) can be fused to a specific artificial repeatdomain which targets the protein to a corresponding DNA sequence in thegenome of the cell. In addition, the DNA recognition specificity ofrepeat domains of the invention can be used to isolate specific DNAsequences. For this, the AvrBs3-like protein can be immobilized to amatrix and interacts with corresponding DNA molecules that contain amatching sequence. Therefore, specific DNA sequences can be isolatedfrom a mixture of DNA molecules.

10) Use of Repeat Domains for the Endonucleolytic Cleavage of DNA

The DNA recognition specificity of the repeat domain can be fused to asuitable restriction endonuclease to specifically cleave DNA. Therefore,the sequence-specific binding of the repeat domain leads to localizationof the fusion protein to few specific sequences, so that theendonuclease specifically cleaves the DNA at the desired location. Bymeans of the recognition of target DNA sequences, unspecific nucleasessuch as FokI can be changed into specific endonucleases analogous towork done with zinc finger nucleases. For example, the optimal distancebetween the two effector DNA target sites would be determined to thatwould be required to support dimerization of two FokI domains. Thiswould be accomplished by analysis of a collection of constructs in whichthe two DNA binding sites are separated by differently sized spacersequences. Using this approach enables one to determine the distancesthat allow nuclease-mediated DNA cleavage to occur and the functionalanalysis of additional effector nucleases that target different DNAsequences. In an alternative approach, a newly developed single-chainFokI dimer (Mino et al. (2009) J Biotechnol 140:156-161) is employed. Inthis approach two FokI catalytic domains are transcriptionally fused toa single repeat domain of the invention. Thus, functionality of acorresponding nuclease no longer relies on intermolecular dimerizationof two FokI domains that are located on two different proteins. Thistype of construct has been used successfully in the context of zincfinger-based DNA binding motifs. Moreover, these methods enable veryspecific cuts at only a few positions in complex DNA-molecules. Thesemethods can amongst other things be used to introduce double-strandbreaks in vivo and selectively incorporate donor DNA at these positions.These methods can also be used to specifically insert transgenes.

11) Construction of Repeat Domains with Custom-Designed Repeat Order

Due to the high similarity between the individual repeat units of arepeat domain, construction of a custom DNA-binding polypeptide asdescribed above might not be feasible through methods involvingtraditional cloning methods. As detailed in this example, a repeatdomain with a repeat unit order that matches a desired DNA-sequence in apromoter of interest, such as the Bs4 promoter (FIGS. 17B, C), isdetermined based on the recognition code of the present invention.Generation of a specific 11.5 repeat unit order was accomplished using“Golden gate” cloning (Engler et al. (2008) PLoS ONE 3:e3647). Asbuilding blocks, we subcloned the N- and C-terminus of Hax3 as well asthe 12 individual repeat units resembling the 11.5 repeat units. Eachbuilding block contained individual flanking BsaI sites (FIG. 18) thatallowed an ordered assembly of the fragments into a custom effectorpolypeptide. The effector (ARTBs4) was correctly assembled from thetotal of 14 fragments into a BsaI-compatible binary vector that allowsAgrobacterium-mediated expression of the custom effector polypeptide asan N-terminally tagged GFP fusion in plant cells (FIG. 18).

12) Use of Effectors as Viral Repressors

The nucleotide binding specificity of the repeat domain can be used todesign effectors that disrupt viral replication in cells. Theseeffectors will exhibit a nucleotide binding specificity targeted tonucleotide sequence in viral origins of replication and other sequencescritical to viral function. No additional protein domains need to befused to these repeat domain proteins in order to block viral function.They act like classical repressors by covering origins of replication orother key sequences, including promoters, enhancers, long terminalrepeat units, and internal ribosome entry sites, by binding and makingthem inaccessible for host or viral factors, including viral encodedRNA-dependent RNA polymerase, nucleocapsid proteins and integrases,which participate in viral replication and function. This type ofstrategy has been used successfully with zinc-finger proteins (Sera(2005) J. Vir. 79:2614-2619; Takenaka et al. (2007) Nucl Acids SymposiumSeries 51:429-430).

Summarizing, the present invention additionally covers isolated nucleicacid molecules to be used in any of the methods of the presentinvention, transformed plants comprising a heterologous polynucleotidestably incorporated in their genome and comprising the nucleotidemolecule described above, preferably operably linked to a promoterelement and/or operably linked to a gene of interest. The transformedplant is preferably a monocot or a dicot. The invention covers alsoseeds of the transformed plants. The invention covers human andnon-human host cells transformed with any of the polynucleotides of theinvention or the polypeptides of the invention. The promoters used incombination with any of the nucleotides and polypeptides of theinvention are preferably tissue specific promoters, chemical-induciblepromoters and promoters inducible by pathogens.

While the present invention can be used in animal and plant systems, onepreferred optional embodiment refers to the use in plant systems. Theterm plant includes plant cells, plant protoplasts, plant cell tissuecultures from which plants can be regenerated, plant calli, plant clumpsand plant cells that are intact in plants or parts of plants such asembryos, pollen, ovules, seed, leaves, flowers, branches, fruits, roots,root tips, anthers and the like. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

Materials and Methods

Bacterial strains and growth conditions. Escherichia coli werecultivated at 37° C. in lysogeny broth (LB) and Agrobacteriumtumefaciens GV3101 at 30° C. in yeast extract broth (YEB) supplementedwith appropriate antibiotics.

Plant material and inoculations. Nicotiana benthamiana plants were grownin the greenhouse (day and night temperatures of 23° C. and 19° C.,respectively) with 16 h light and 40 to 60% humidity. Mature leaves offive- to seven-week-old plants were inoculated with Agrobacterium usinga needleless syringe as described previously (S1). Inoculated plantswere transferred to a Percival growth chamber (Percival Scientific) with16 h light, 22° C. and 18° C. night temperature.

Construction of artificial effectors. The construction of effectors withmodified repeat region was based on ligation of Esp3I (Fermentas)restriction fragments. Esp3I cuts outside of its recognition sequenceand typically once per repeat. To construct a GATEWAY(Invitrogen)-compatible ENTRY-vector for generation of effectors of theinvention, the N- and C-termini of hax3 were amplified by PCR using aproof reading polymerase (HotStar HiFidelity Polymerase Kit; Qiagen),combined by SOE (splicing by overlap extension)-PCR and inserted intopCR8/GW/TOPO resulting in a hax3-derivative with 1.5 repeat units(pC3SE26; first repeat=NI; last half repeat=NG). A 1 bp frame-shiftpreceding the start codon was inserted by site-directed mutagenesis toallow in frame N-terminal fusions using GATEWAY recombination(Invitrogen) resulting in pC3SEIF. Single repeat units were amplifiedfrom TAL effectors using a forward primer binding to most repeat unitsand repeat-specific reverse primers. Both primers included the naturallypresent Esp3I sites. To avoid amplification of more than one repeat,template DNA was digested with Esp3I prior to the PCR reaction.PCR-products were digested with Esp3I and cloned into Esp3I-digestedpC3SE26 yielding Hax3-derivatives with 2.5 repeat units where a singlerepeat can be excised with Esp3I (HD-repeat=repeat 5 of Hax3;NI-repeat=repeat 11 of Hax3; NG-repeat=repeat 4 of Hax4; NN-repeat=G₁₃Nmutant of repeat 4 of Hax4). The ArtHD effector backbone constructconsists of the N- and C-terminus of Hax3 with the last half repeatmutated into a HD-repeat. The resulting construct was restricted byEsp3I and dephosphorylated. DNA fragments encoding repeat units wereexcised with Esp3I from pC3SE26-derivatives containing a singleHD-repeat and purified via agarose gels. Ligation was performed using amolar excess of insert to vector to facilitate concatemer ligation andtransformed into E. coli. The number of repeat units was determined inrecombinant plasmids using StuI and HincII. ArtX1-3 effectors with arandom combination of repeat types were generated by isolating DNAfragments encoding repeat units as described above from cloned singleNI-, HD-, NN-, and NG-repeat units (specificities for A, C, G/A, and T,respectively). The fragments were added in equal molar amounts each tothe concatemer ligation reaction with vector pC3SEIF. Plasmidscontaining effectors of the invention with 12.5 repeat units were chosenfor subsequent analysis. Effectors were cloned by GATEWAY-recombination(Invitrogen) into pGWB6 (S2) for expression of N-terminal GFP-effectorfusions. Oligonucleotide sequences are available upon request. Allconstructs were sequenced.

GUS reporter constructs. The minimal Bs4 promoter was amplified by PCRand inserted into pENTR/D-TOPO (Invitrogen) with target DNA boxes at the5′ end (S3; FIG. S5). Promoter derivatives were cloned into pGWB3 (S2)containing a promoterless uidA gene.

Construction of hax2-transgenic A. thaliana. hax2 was cloned undercontrol of the inducible alcA promoter from Aspergillus nidulans into aGATEWAY-compatible derivative of the binary T-DNA vector binSRNACatN(Zeneca Agrochemicals) containing the 35S-driven alcR ethanol-dependentregulator gene and a nptII selection marker. AlcR drivesethanol-dependent induction of the alcA promoter (S4). T-DNA containingthese genes was transformed into A. thaliana Col-0 via A. tumefaciensusing floral dip inoculation (S5). Transformants were selected askanamycin-resistant plants on sterile medium.

Construction of ARTBs4, an artificial effector. “Golden gate” cloning(Engler et al. (2008) PLoS ONE 3:e3647) was used to assemble effectorswith 11.5 specifically ordered repeat units. The N- and C-terminus ofHax3 and 12 individual repeat units resembling the 11.5 repeat unitswere subcloned. Each building block contained individual flanking BsaIsites that allowed an ordered assembly of the fragments into anartificial effector. For the targeted assembly of effectors with anydesired repeat composition, the building block repertoire of repeatunits was expanded. To allow for target specificity to any of the fournatural bases (A, C, G, and T) in DNA, four different repeat types werechosen, based on the amino acids 12 and 13 per repeat unit. The fourrepeat types and their specificities are: NI=A; HD=C; NG=T, NN=G or A.To generate a universally applicable assembly kit, four unitscorresponding to each of the four repeat unit types were cloned withflanking BsaI sites for each of the 12 repeat positions. The sum of 48building blocks resembles a library that can be used to assembleeffectors with 11.5 repeat units with any composition of the four repeatunit types.

β-Glucuronidase (GUS) assays. For transient GUS assays Agrobacteriumstrains delivering effector constructs and GUS reporter constructs weremixed 1:1, and inoculated into Nicotiana benthamiana leaves with anOD₆₀₀ of 0.8. Two leaf discs (0.9 cm diameter) were sampled two dayspost infiltration (dpi) and quantitative GUS activity was determinedusing 4-methyl-umbelliferyl-β-D-glucuronide (MUG), as describedpreviously (Si). Proteins were quantified using Bradford assays(BioRad). Data correspond to triplicate samples from different plants.For qualitative GUS assays, leaf discs were sampled 2 dpi, incubated inX-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) staining solution(S3), destained in ethanol, and dried. Experiments were performed atleast twice with similar results.

Expression of hax2, hax3, and hax4. hax2, hax3, and hax4 were expressedin planta under control of the constitutive cauliflower mosaic virus 35Spromoter using pAGH2, pAGH3, and pAGH4 (S6).

DNaseI footprinting. DNaseI footprinting was performed as described (S7)with the following modifications: Fluorescently labeled PCR products ofBs3 and Bs3-E promoter DNA were generated using plasmidspCRBluntII-TOPO::FPBs3 (Bs3 promoter fragment from −211 to +108) andpCRBluntII-TOPO::FPBs3-E (Bs3-E promoter fragment from −224 to +108),respectively, as template and Phusion DNA polymerase (Finnzymes).Fluorescently labeled PCR product of UPA20-ubm-r16 promoter DNA wasgenerated using plasmid pCRBluntII-TOPO::FPU20-ubm-r16 (UPA20 promoterfragment from −213 to +86 containing the ubm-r16 mutation (S7) astemplate and Phusion DNA polymerase (Finnzymes). PlasmidspCRBluntII-TOPO::FPBs3, pCRBluntII-TOPO::FPBs3-E andpCRBluntII-TOPO::FPU20-ubm-r16 were sequenced, using the ThermoSequenase Dye Primer Manual Cycle Sequencing Kit (USB) according to themanufacturer's instructions. An internal Gene Scan-500 LIZ Size Standard(Applied Biosystems) was used to determine the DNA fragment size.

Example 2 Identification of a TAL Repeat Unit That Binds to GNucleotides

The DNA binding domain of TAL effectors is composed of tandem-arranged34-amino acid repeat units. The amino acid sequences of the repeat unitsare mostly conserved, except for two adjacent highly variable residues(HVRs) at positions 12 and 13 that define DNA target specificity (Bochet al. (2009) Science 326:1509-1512; Moscou & Bogdanove (2009) Science326:1501). Functional analysis identified HVR motifs that bindpreferentially to A (NI), C(HD), T (NG, IG) or equally well to G and A(NN) (Boch et al. (2009) Science 326:1509-1512). Bioinformatic analysisrevealed HVRs that in the given promoter-TAL effector interactions matchspecifically to G (Moscou & Bogdanove (2009) Science 326:1501). Howeverthis, analysis was based on a single (HN & NA) or two (NK) interactionsites. In our view the number of interaction sites is too low to makereliable conclusions on the HVR specificity. Yet, these HVRs can beconsidered as suitable candidates that may mediate specific binding toG.

In order to clarify the target specificity of HVRs with unknownspecificity we made use of the well-characterized interaction betweenAvrBs3 and the UPA box in the Bs3 promoter. Using site directedmutagenesis we replaced the HVR NI in the 5^(th) and the 6^(th) repeatunit by NK resulting in AvrBs3-NK_(5/6). In the wildtype Bs3 promoterthe NI residues of the 5^(th) and the 6^(th) repeat both match to Anucleotides. Using site-directed mutagenesis we replaced the two Anucleotides in the Bs3 promoter by two C, G and T nucleotides. Thewildtype Bs3 promoter and the three promoter mutants were fused to anuidA reporter gene and tested via Agrobacterium tumefaciens transientexpression in combination with either wildtype AvrBs3 or AvrBs3-NK_(5/6)in Nicotiana benthamiana leaves. GUS assays revealed thatAvrBs3-NK_(5/6) activated the GUS reporter only in combination with the“GG” Bs3 promoter mutant while AvrBs3 activated only the Bs3 wildtypepromoter construct.

Our analysis suggests that NK pairs specifically to G and thus providesan option to generate more specific repeat arrays and also tospecifically target G-rich target sequences.

Example 3 Method for Generation of Designer Effectors via Golden GateCloning

The DNA binding domain of TAL effectors is composed of tandem-arranged34-amino acid repeat units. The amino acid sequences of the repeat unitsare mostly conserved, except for two adjacent highly variable residues(HVRs) at positions 12 and 13 that define DNA target specificity (Bochet al. (2009) Science 326:1509-1512; Moscou & Bogdanove (2009) Science326:1501). Different HVR motifs bind with different levels ofspecificity to individual A, C, G or T nucleotides. Importantly,statistical analysis suggests that tandem arranged repeat units do notto interfere with the specificity of adjacent units (Moscou & Bogdanove(2009) Science 326:1501). Thus modular assembly of repeat units withpre-characterized specificities is likely to provide an efficient wayfor generation of DNA-recognition modules with desired DNA specificity.

However, the generation of DNA constructs that encode desired repeatdomains is challenging due to the fact that the repeat units are almostidentical. In the past we have used chemical synthesis to generateeffectors genes that encode 17.5 repeat units with the desired HVRcomposition. To maximize the differences between repeat units at the DNAlevel we exploited the degeneracy of the genetic code. Thecodon-optimized sequence of the 17.5 repeat unit encoding DNA sequencewas, in contrast to the corresponding TAL effector wildtype gene,PCR-amplifiable and amenable to PCR-based mutagenesis. Our findings alsodemonstrate that chemical synthesis of effector repeat domains isgenerally feasible. However, chemical synthesis does not allow rapid andcost-efficient generation of multiple effectors with desired HVRcomposition. Furthermore this approach will most likely not allowgeneration of repeat domains with 20 or more repeat units.

The recently developed “Golden-Gate cloning” provides an alternativeapproach for generation of repeat unit arrays of desired composition.The strategy is based on the use of type IIS restriction enzymes, whichcut outside of their recognition sequence. We will work with the typeIIS enzyme BsaI, which creates a 4-bp sticky end. Due to the fact, thatrecognition and cleavage site are separated in type IIS enzymes we cangenerate by BsaI restriction in principle 256 (4⁴) different sticky endswhich provides the basis for multi fragment ligations. With properdesign of the cleavage sites, two or more fragments cut by type IISrestriction enzymes can be ligated into a product lacking the originalrestriction site (Engler et al. (2008) PLoS ONE 3:e3647; Engler et al.(2009) PLoS ONE 4:e5553).

However in practice there are two limitations to this method. Due toexonuclease activity in some reactions, single stranded overhanging DNAsticky ends are reduced from four to three bases, effectively making thenumber of compatible sticky ends only 16 (2⁴). Secondly, the efficiencyof the ligation reactions decreases precipitously with large numbers ofinserts, such as would be needed to create an effector with 17.5 repeatunits as typically found in naturally occurring functional TALeffectors. To circumvent these limitations, we have designed a two-stageligation process that allows the effective production of effectors of20, 30, 40 or more repeat units.

The basis for our “repeat-array building kit” is a set of “insertplasmids” that contain individual repeat units (one repeat unit perplasmid), “intermediate vectors” that contain repeat domains consistingof sets of 10 repeat units, and one “acceptor vector” that contains theN- and C-terminal non-repeat region of a TAL effector. All repeat unitsare designed in such a way that the BsaI recognition sites flank theinsert in the insert plasmids.

To simplify the explanation of the multi-fragment ligation we defineherein the different ends of the repeat unit genes with upper caseletters (instead of the sequence overhang of the sticky end) andindicate their orientation (N- or C-terminus of the repeat unit) with Nor C in square brackets (e.g. A[C]). The insert plasmid containing the1^(st) repeat unit gene is designed in such a way that BsaI treatmentcreates A[N] and B[C] termini. The 2^(nd) repeatunit gene has B[N] andC[C] termini upon BsaI cleavage, while BsaI cleavage of the insertplasmid with the 3^(rd) repeat unit gene results in C[N] and D[C]termini, and so on. Since only compatible ends can be fused, the B[C]terminus of the 1^(st) repeat unit gene will fuse specifically to theB[N] terminus of the 2^(nd) repeat unit gene. Similarly the C[C]terminus of the 2^(nd) repeat unit gene will ligate specifically to theC[N] terminus of the 3^(rd) repeat unit gene and so on.

BsaI digestion releases the repeat units with 4-bp sticky overhangs thatare compatible only with the designed adjacent repeat units. The BsaIrecognition site itself remains in the cleaved insert plasmid vector andthe released insert has no BsaI recognition site. The repeat units arejoined together in the order specified by the overhanging ends in acut-ligation reaction (cleavage and ligation running simultaneously).Due to the simultaneous action of BsaI and ligase the religation ofrepeat units into the insert donor vector is avoided since this restoresthe BsaI recognition site. By contrast the desired ligation productslack the BsaI recognition sites. This experimental design makes thiscloning procedure highly efficient.

To generate effectors that are designed to recognize specific basesequences, four variants are made for each repeat unit position. Thesevariants are individual repeat units with specific nucleotiderecognition specificity, (e.g. HD residues at position 12 and 13 forrecognition of a C base, NI for A, and so on). The variant for eachposition is made with the appropriate sticky ends for each repeat unit,for example A[N] and B[C] termini for repeat unit 1, such that there arefour possible insert plasmids for repeat unit one, chosen based on thedesired DNA recognition. There are four variants for repeat unit 2, withdifferent nucleotide recognition specificity and B[N] and C[C] termini,and so on for each repeat position

Ligations are carried out in two stages. In the first stage, 10 repeatunits are combined into an intermediate vector. Different sets of 10repeat units can be combined in intermediate vectors. Intermediatevector 1 contains repeat units 1-10, intermediate vector 2 containsrepeat units 11-20 and so on. In the second stage, separately assembled10 repeat units are combined into acceptor vectors. The acceptor vectoralso contains the N- and C-terminal non-repeat areas of the effector,such that a complete effector comprised of 10, 20, 30 40 or othermultiples of 10 repeat units is assembled in the final construct. Theintermediate vector has BsaI sites in the insert for introducing the 10repeat unit fragments and also has flanking BpiI sites in the flankingvector sequence. BpiI is another type IIS enzyme with a recognition sitedistinct from BsaI. Using BsaI, the 10 repeat units are first assembledinto the “intermediate vector” and using BpiI the assembled 10 mers arereleased as one fragment. This fragment is ligated in a BpiI cut-ligasereaction with the acceptor vector, which contains BpiI sites between theN- and C-terminal non-repeat areas of the TAL effector. In this caseonly 2-4 inserts are ligated into the acceptor vector. This allows tomake each ligation highly specific and to assemble easily 40 and morerepeat units.

The acceptor vector in which the repeat unit array is finally cloned,represents a GATEWAY Entry clone and thus allows recombination-basedtransfer of the effector into any desired expression construct.Currently the acceptor vector is designed to generate a TAL-typetranscription factor. However, with few modifications the acceptorvector allows also fusions of the repeat array to the FokI endonucleaseor other desired functional domains.

A schematic of this method is provided in FIG. 19A-D.

Example 4 Production and Testing of Target DNA-Specific Nucleases

Fusion proteins comprising a repeat domain of the invention thatrecognizes a target DNA sequence and a FokI nuclease(“TAL-type-nucleases”) are produced as described by any of the methoddisclosed herein or known in the art. The fusion proteins are tested fornuclease activity by incubation with corresponding target DNA. Therepeat domain DNA target site is cloned into the multiple cloning siteof a plasmid vector (e.g., bluescript). As negative controls, either an“empty vector” that contains no TAL-nuclease target site or clonedtarget sites with mutations are used. Before treatment of the DNAsubstrate with the TAL-type nuclease, the vector is linearized bytreatment with a suitable standard endonuclease that cleaves in thevector backbone. This linearized vector is incubated with in vitrogenerated repeat domain-FokI nuclease fusion proteins and the productsanalyzed by agarose gel electrophoresis. The detection of two DNAfragments in gel electrophoresis is indicative for specific nucleasemediated cleavage. By contrast, the negative controls that do notcontain a target site that is recognized by repeat domain are unaffectedby treatment with the repeat domain-FokI nuclease fusion protein.DNA-driven, cell-free systems for in vitro gene expression and proteinsynthesis are used to generate repeat domain-FokI nuclease fusionproteins (e.g. T7 High-Yield Protein Expression System; Promega). To usesuch systems, repeat domain-FokI nuclease fusion protein nucleotidesequences are cloned in front of a T7 RNA polymerase. Such fusionproteins that are produced via in vitro transcription and translationare used in DNA cleavage assays without further purification.

Example 5 Determination of Additional Recognition Specificites

Further experiments were conducted essentially as described hereinaboveto determine the recognition specificities of additional amino acidpairs in the hypervariable region. DNA binding domains were constructedusing Golden Gate Cloning as described in Example 3. The experimentsconducted and the experimental results obtained are provided in FIGS.20-27 and their respective figure legends.

From these experiments, the recognition specificity for the amino acidsfound at positions 12 and 13 in a repeat unit and the base pair in thetarget DNA sequence were determined for the following amino acid pairs:

-   -   NH for recognition of G/C    -   NP for recognition of A/T or C/G or T/A    -   NT for recognition of A/T or G/C    -   HN for recognition of A/T or G/C    -   SH for recognition of G/C    -   SN for recognition of G/C and    -   IS for recognition of A/T.

It is recognized that the recognition specificities set forth in thisExample can be used in the methods of the present invention. It isfurther recognized that the recognition specificities set forth in thisExample can be used to produce compositions of the present invention,such as, for example, polypeptides and DNA. Preferably, the recognitionspecificities set forth in this Example are used in such methods or toproduce such compositions in combination with any of the otherrecognition specificities disclosed herein.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. Additionally, each of thefollowing patent applications is hereby herein incorporated referencedin its entirety: DE 10 2009 004 659.3 filed Jan. 12, 2009, EP 09165328filed Jul. 13, 2009, and U.S. 61/225,043 filed Jul. 13, 2009.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method for producing a polypeptide that selectively recognizes atleast one base pair in a target DNA sequence, the method comprisingsynthesizing a polypeptide comprising a repeat domain, wherein therepeat domain comprises at least one repeat unit derived from atranscription activator-like (TAL) effector, wherein the repeat unitcomprises a hypervariable region which determines recognition of a basepair in the target DNA sequence, wherein the repeat unit is responsiblefor the recognition of one base pair in the DNA sequence, and whereinthe hypervariable region comprises a member selected from the groupconsisting of: (a) HD for recognition of C/G; (b) NI for recognition ofA/T; (c) NG for recognition of T/A; (d) NS for recognition of C/G or A/Tor T/A or G/C; (e) NN for recognition of G/C or A/T; (f) IG forrecognition of T/A; (g) N for recognition of C/G or T/A; (h) HG forrecognition of C/G or T/A; (i) H for recognition of T/A; (j) NK forrecognition of G/C; (k) NH for recognition of G/C; (l) NP forrecognition of A/T or C/G or T/A; (m) NT for recognition of A/T or G/C;(n) HN for recognition of A/T or G/C; (o) SH for recognition of G/C; (p)SN for recognition of G/C; and (q) IS for recognition of A/T; whereinthe repeat domain comprises at least one repeat unit which comprises ahypervariable region comprising (k), (l), (m), (n), (o), (p), or (q). 2.(canceled)
 3. The method of claim 1, wherein the hypervariable regioncorresponds to amino acids 12 and 13 in the repeat unit.
 4. The methodof claim 1, wherein the repeat domain comprises 1.5 to 40.5 repeatunits.
 5. The method of claim 1, wherein the repeat domain comprises11.5 to 33.5 repeat units.
 6. The method of claim 1, wherein thepolypeptide further comprises at least one additional domain that isoperably linked to the repeat domain.
 7. The method of claim 6, whereinthe additional domain comprises a bacterial, viral, fungal, oomycete,human, animal, plant, or artificial protein, or part thereof.
 8. Themethod of claim 6, wherein the additional domain comprises a protein orfunctional part or domain thereof, that is capable of modifying DNA orRNA.
 9. The method of claim 6, wherein the additional domain comprises aprotein or functional part or domain thereof selected from the groupconsisting of: a transcription activator, a transcription repressor, aresistance-mediating protein, a nuclease, a topoisomerase, a ligase, anintegrase, a recombinase, a resolvase, a methylase, an acetylase, ademethylase, and a deacetylase.
 10. The method of claim 1, wherein therepeat domain of the polypeptide is synthesized by expressing a DNAsequence encoding the polypeptide and where the DNA sequence encodingthe polypeptide is assembled by preassembling the repeat units in one ormore target vectors that can subsequently be assembled into a finalvector comprising the DNA sequence encoding the polypeptide.
 11. Themethod of claim 1, wherein the repeat unit comprises 30 to 40 aminoacids.
 12. The method of claim 11, wherein the repeat unit comprises 33,34, 35 or 39 amino acids.
 13. The method of claim 1, wherein thepolypeptide recognizes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 base pairs in the target DNA sequence. 14.The method of claim 13, wherein the polypeptide recognizes all of thebase pairs in the target DNA sequence.
 15. The method of claim 13,wherein the polypeptide is capable of binding to the target DNAsequence.
 16. A polypeptide produced by the method of claim
 1. 17. Thepolypeptide of claim 16, wherein the polypeptide is not naturallyoccurring.
 18. A polynucleotide molecule comprising a coding sequencefor the polypeptide produced by the method of claim
 1. 19. Thepolynucleotide molecule of claim 18, wherein the polynucleotide moleculeis not naturally occurring.
 20. An expression cassette comprising apromoter operably linked to the polynucleotide molecule of claim
 18. 21.A non-human host cell comprising the expression cassette of claim 20.22. The host cell of claim 21, wherein the host cell is a bacterialcell, a fungal cell, an animal cell, or a plant cell.
 23. A transformed,non-human organism comprising the expression cassette of claim
 20. 24.The transformed organism of claim 23, wherein the organism is a fungus,an animal, or a plant.
 25. A method for selectively recognizing a basepair in a DNA sequence by a polypeptide, the method comprisingconstructing a polypeptide comprising a repeat domain, wherein therepeat domain comprises at least one repeat unit derived from a TALeffector, wherein the repeat unit comprises a hypervariable region whichdetermines recognition of a base pair in the DNA sequence, wherein therepeat unit is responsible for the recognition of one base pair in theDNA sequence, and wherein the hypervariable region comprises a memberselected from the group consisting of: (a) HD for recognition of C/G;(b) NI for recognition of A/T; (c) NG for recognition of T/A; (d) NS forrecognition of C/G or A/T or T/A or G/C; (e) NN for recognition of G/Cor A/T; (f) IG for recognition of T/A; (g) N for recognition of C/G orT/A; (h) HG for recognition of C/G or T/A; (i) H for recognition of T/A;(j) NK for recognition of G/C; (k) NH for recognition of G/C; (l) NP forrecognition of A/T or C/G or T/A; (m) NT for recognition of A/T or G/C;(n) HN for recognition of A/T or G/C; (o) SH for recognition of G/C; (p)SN for recognition of G/C; and (q) IS for recognition of A/T; whereinthe repeat domain comprises at least one repeat unit which comprises ahypervariable region comprising (k), (l), (m), (n), (o), (p), or (q).26. (canceled)
 27. The method of claim 25, wherein the hypervariableregion corresponds to amino acids 12 and 13 in the repeat unit.
 28. Themethod of claim 25, wherein the repeat domain comprises 1.5 to 40.5repeat units.
 29. The method of claim 25, wherein the repeat domaincomprises 11.5 to 33.5 repeat units.
 30. The method of claim 25, whereinthe polypeptide further comprises at one additional domain that isoperably linked to the repeat domain.
 31. The method of claim 25,wherein the repeat unit comprises 30 to 40 amino acids.
 32. The methodof claim 31, wherein the repeat unit comprises 33, 34, 35 or 39 aminoacids.
 33. The method of claim 25, wherein the repeat domain comprisingrepeat units is inserted in a bacterial, viral, fungal, oomycete, human,animal or plant polypeptide to achieve a targeted recognition andpreferably binding of one or more specified base pairs in a DNAsequence, and optionally wherein the repeat unit is derived from therepeat domains of AvrBs3-like effectors which are further optionallymodified in order to obtain a pre-selected specific activity to one ormore base pairs in a DNA sequence.
 34. The method of claim 25, whereinthe repeat domain comprising the repeat unit is contained in apolypeptide controlling the transcription of a gene, optionally intranscription activator or repressor proteins, optionally in AvrBs3-likeproteins, e.g. in AvrBs3 or Hax effector proteins.
 35. The method ofclaim 25, wherein the N-terminal region of a repeat domain confers arecognition specificity for a T/A 5′ of the recognition specificity ofthe repeat unit.
 36. The method of claim 25, wherein the base pair inthe DNA sequence is inserted into an expression control element combinedwith a gene, the expression control element being targeted by atranscription control protein comprising the hypervariable region in therepeat unit recognizing the base pair located in the expression controlelement to specifically control the expression of the gene, wherein theexpression control element is preferably a promoter.
 37. The method ofclaim 36, wherein the gene is a resistance mediating gene in order toobtain a disease resistant organism, the expression control elementbeing optionally the target sequence for an AvrBs3-like effectorprotein.
 38. A method of modulating expression of a target gene in acell, wherein cells are provided which contain a polypeptide wherein thepolypeptide comprises a repeat domain, wherein the repeat domaincomprises at least one repeat unit derived from a TAL effector, whereinthe repeat unit comprises a hypervariable region which determinesrecognition of a base pair in a DNA sequence, wherein the repeat unit isresponsible for the recognition of one base pair in the DNA sequence,and wherein the hypervariable region comprises a member selected fromthe group consisting of: (a) HD for recognition of C/G; (b) NI forrecognition of A/T; (c) NG for recognition of T/A; (d) NS forrecognition of C/G or A/T or T/A or G/C; (e) NN for recognition of G/Cor A/T; (f) IG for recognition of T/A; (g) N for recognition of C/G orT/A; (h) HG for recognition of C/G or T/A; (i) H for recognition of T/A;(j) NK for recognition of G/C; (k) NH for recognition of G/C; (l) NP forrecognition of A/T or C/G or T/A; (m) NT for recognition of A/T or G/C;(n) HN for recognition of A/T or G/C; (o) SH for recognition of G/C; (p)SN for recognition of G/C; and (q) IS for recognition of A/T; whereinthe repeat domain comprises at least one repeat unit which comprises ahypervariable region comprising (k), (l), (m), (n), (o), (p), or (q).39. (canceled)
 40. A polypeptide comprising a repeat domain, wherein therepeat domain comprises at least one repeat unit derived from a TALeffector, wherein the repeat unit comprises a hypervariable region whichdetermines recognition of a base pair in a DNA sequence, wherein therepeat unit is responsible for the recognition of one base pair in theDNA sequence, and wherein the hypervariable region comprises a memberselected from the group consisting of: (a) HD for recognition of C/G;(b) NI for recognition of A/T; (c) NG for recognition of T/A; (d) NS forrecognition of C/G or A/T or T/A or G/C; (e) NN for recognition of G/Cor A/T; (f) IG for recognition of T/A; (g) N for recognition of C/G orT/A; (h) HG for recognition of C/G or T/A; (i) H for recognition of T/A;(j) NK for recognition of G/C; (k) NH for recognition of G/C; (l) NP forrecognition of A/T or C/G or T/A; (m) NT for recognition of A/T or G/C;(n) HN for recognition of A/T or G/C; (o) SH for recognition of G/C; (p)SN for recognition of G/C; and (q) IS for recognition of A/T; whereinthe repeat domain comprises at least one repeat unit which comprises ahypervariable region comprising (k), (l), (m), (n), (o), (p), or (q).41. (canceled)
 42. A polynucleotide molecule comprising a codingsequence for the polypeptide of claim
 40. 43. A DNA which is modified toinclude a at least one base pair located in a target DNA sequence sothat the base pair can be specifically recognized by a polypeptidecomprising a repeat domain, wherein the repeat domain comprises at leastone repeat unit derived from a TAL effector, wherein the repeat unitcomprises a hypervariable region which determines recognition of a basepair in the DNA sequence, wherein the repeat unit is responsible for therecognition of one base pair in the DNA sequence, and wherein the basepair is selected from the group consisting of: (a) G/C for recognitionof NH; (b) A/T or C/G or T/A for recognition of NP; (c) A/T or G/C forrecognition of NT; (d) A/T or G/C for recognition of HN; (e) G/C forrecognition of SH; (f) G/C for recognition of SN; and (g) A/T forrecognition of IS.
 44. The DNA of claim 43, wherein at least oneadditional base pair is selected from the group consisting of: (h) C/Gfor recognition by HD; (i) A/T for recognition by NI; (j) T/A forrecognition by NG; (k) CT or A/T or T/A or G/C for recognition by NS;(l) G/C or A/T for recognition by NN; (m) T/A for recognition by IG; (n)C/G or T/A for recognition by N; (o) T/A for recognition by HG; (p) T/Afor recognition by H; and (q) G/C for recognition by NK.
 45. The DNA ofclaim 43, wherein the base pair is located in a promoter or other generegulatory sequence.
 46. The DNA of claim 43, wherein the DNA is notnaturally occurring.
 47. A vector comprising the DNA of claim
 43. 48. Anon-human host cell comprising the DNA of claim
 43. 49. The host cell ofclaim 43, wherein the host cell is a bacterial cell, a fungal cell, ananimal cell, or a plant cell.
 50. A transformed, non-human organismcomprising the DNA of claim
 43. 51. The transformed organism of claim50, wherein the organism is a fungus, an animal, or a plant.
 52. Amethod for producing a DNA comprising a target DNA sequence that isselectively recognized by a polypeptide comprising a repeat domain,wherein the repeat domain comprises at least one repeat unit derivedfrom a TAL effector, wherein the repeat unit comprises a hypervariableregion which determines recognition of a base pair in the target DNAsequence, wherein the repeat unit is responsible for the recognition ofone base pair in the target DNA sequence, the method comprisingsynthesizing a DNA comprising a base pair that is capable of beingrecognized by the repeat unit, and wherein the base pair is selectedfrom the group consisting of: (a) C/G for recognition by HD; (b) A/T forrecognition by NI; (c) T/A for recognition by NG; (d) CT or A/T or T/Aor G/C for recognition by NS; (e) G/C or A/T for recognition by NN; (f)T/A for recognition by IG; (g) C/G or T/A for recognition by N; (h) T/Afor recognition by HG; (i) T/A for recognition by H; (j) G/C forrecognition by NK; (k) G/C for recognition of NH; (l) A/T or C/G or T/Afor recognition of NP; (m) A/T or G/C for recognition of NT; (n) A/T orG/C for recognition of HN; (o) G/C for recognition of SH; (p) G/C forrecognition of SN; and (q) A/T for recognition of IS and wherein therepeat domain comprises at least one repeat unit which comprises ahypervariable region comprising at least one member selected from thegroup consisting of (i) NH for recognition of G/C; (ii) NP forrecognition of A/T or C/G or T/A; (iii) NT for recognition of A/T orG/C; (iv) HN for recognition of A/T or G/C; (v) SH for recognition ofG/C; (vi) SN for recognition of G/C; and (vii) IS for recognition ofA/T.
 53. (canceled)